To see the other types of publications on this topic, follow the link: Enzyme mutations.
Journal articles on the topic 'Enzyme mutations'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Enzyme mutations.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Coates, Talmage L., Naomi Young, Austin J. Jarrett, Connor J. Morris, James D. Moody, and Dennis Della Corte. "Current computational methods for enzyme design." Modern Physics Letters B 35, no. 09 (February 12, 2021): 2150155. http://dx.doi.org/10.1142/s0217984921501554.
Full textAbstract:
Computational enzyme design has made great strides over the last five years. Traditional methods of enzyme design require synthesis and evaluation of many mutations. Computational enzyme design has emerged as a powerful tool to predict how specific mutations modify a protein’s activity, stability, and/or selectivity. Such computational approaches can evaluate many mutations and reduce the load of in vitro work by identifying mutations likely to accomplish design objectives. Computational approaches can explore mutational spaces inaccessible in traditional mutagenesis. Computational methods reduce cost and time compared with experimental approaches. We review the efficacy and key differences of computational enzyme design methods as published in recent studies. The included articles used computational methods to design enzymes, were published no earlier than 2015, met design objectives, and verified results in vitro.
2
Perutz, M. F. "Mutations make enzyme polymerize." Nature 385, no. 6619 (February 1997): 773–75. http://dx.doi.org/10.1038/385773a0.
Full text
3
Klesmith, Justin R., John-Paul Bacik, Emily E. Wrenbeck, Ryszard Michalczyk, and Timothy A. Whitehead. "Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning." Proceedings of the National Academy of Sciences 114, no. 9 (February 14, 2017): 2265–70. http://dx.doi.org/10.1073/pnas.1614437114.
Full textAbstract:
Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.
4
Salverda, Merijn L. M., Jeroen Koomen, Bertha Koopmanschap, Mark P. Zwart, and J. Arjan G. M. de Visser. "Adaptive benefits from small mutation supplies in an antibiotic resistance enzyme." Proceedings of the National Academy of Sciences 114, no. 48 (November 13, 2017): 12773–78. http://dx.doi.org/10.1073/pnas.1712999114.
Full textAbstract:
Populations with large mutation supplies adapt via the “greedy” substitution of the fittest genotype available, leading to fast and repeatable short-term responses. At longer time scales, smaller mutation supplies may in theory lead to larger improvements when distant high-fitness genotypes more readily evolve from lower-fitness intermediates. Here we test for long-term adaptive benefits from small mutation supplies using in vitro evolution of an antibiotic-degrading enzyme in the presence of a novel antibiotic. Consistent with predictions, large mutant libraries cause rapid initial adaptation via the substitution of cohorts of mutations, but show later deceleration and convergence. Smaller libraries show on average smaller initial, but also more variable, improvements, with two lines yielding alleles with exceptionally high resistance levels. These two alleles share three mutations with the large-library alleles, which are known from previous work, but also have unique mutations. Replay evolution experiments and analyses of the adaptive landscape of the enzyme suggest that the benefit resulted from a combination of avoiding mutational cohorts leading to local peaks and chance. Our results demonstrate adaptive benefits from limited mutation supplies on a rugged fitness landscape, which has implications for artificial selection protocols in biotechnology and argues for a better understanding of mutation supplies in clinical settings.
5
Puranen, T. J., M. H. Poutanen, H. E. Peltoketo, P. T. Vihko, and R. K. Vihko. "Site-directed mutagenesis of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1." Biochemical Journal 304, no. 1 (November 15, 1994): 289–93. http://dx.doi.org/10.1042/bj3040289.
Full textAbstract:
Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.
6
NJÅLSSON, Runa, Katarina CARLSSON, Vikas BHANSALI, Jia-Li LUO, Lennart NILSSON, Rudolf LADENSTEIN, Mary ANDERSON, Agne LARSSON, and Svante NORGREN. "Human hereditary glutathione synthetase deficiency: kinetic properties of mutant enzymes." Biochemical Journal 381, no. 2 (July 6, 2004): 489–94. http://dx.doi.org/10.1042/bj20040114.
Full textAbstract:
Patients with hereditary glutathione synthetase deficiency suffer from haemolytic anaemia, 5-oxoprolinuria, metabolic acidosis, recurrent bacterial infections and various degrees of central nervous system dysfunction. To investigate the molecular basis of the mutations associated with this disease, seven naturally occurring missense mutations [L188P (Leu188→Pro), D219A, D219G, Y270C, Y270H, R283C and P314L] were expressed using a His-tagged, Escherichia coli-based expression system. Effects of the mutations on kinetic properties, including negative co-operative binding of γ-glutamyl substrate, were evaluated. The mutation P314L did not have any major effect on these parameters and was classified as a neutral mutation. The remaining mutations decreased Vmax to 2–27% of wild-type activity. Negative co-operativity for γ-gluABA (L-γ-glutamyl-L-α-aminobutyric acid) was abolished in five mutant recombinant enzymes, whereas for one mutant enzyme, this co-operativity changed from negative to positive. The structural consequences of the mutations were interpreted on the basis of the known structure of the wild-type enzyme.
7
Yanase, Michiyo, Hiroki Takata, Kazutoshi Fujii, Takeshi Takaha, and Takashi Kuriki. "Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase." Applied and Environmental Microbiology 71, no. 9 (September 2005): 5433–39. http://dx.doi.org/10.1128/aem.71.9.5433-5439.2005.
Full textAbstract:
ABSTRACT The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.
8
Bebenek, Anna, Geraldine T. Carver, Holly Kloos Dressman, Farid A. Kadyrov, Joseph K. Haseman, Vasiliy Petrov, William H. Konigsberg, Jim D. Karam, and John W. Drake. "Dissecting the Fidelity of Bacteriophage RB69 DNA Polymerase: Site-Specific Modulation of Fidelity by Polymerase Accessory Proteins." Genetics 162, no. 3 (November 1, 2002): 1003–18. http://dx.doi.org/10.1093/genetics/162.3.1003.
Full textAbstract:
Abstract Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3′ exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol+ Exo+) enzyme, an exonuclease-deficient mutator variant (Pol+ Exo-), mutator variants with substitutions at Tyr567 in the polymerase active site (PolM Exo+), and the double mutator PolM Exo-. Comparing the mutational spectra of the Pol+ Exo- and Pol+ Exo+ enzymes revealed the patterns and efficiencies of proofreading, while Tyr567 was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.
9
Patel, Meha P., Bartlomiej G. Fryszczyn, and Timothy Palzkill. "Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases." Antimicrobial Agents and Chemotherapy 59, no. 11 (August 17, 2015): 6741–48. http://dx.doi.org/10.1128/aac.00618-15.
Full textAbstract:
ABSTRACTThe widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of β-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active site-associated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 β-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extended-spectrum β-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of β-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levelsin vivoand protein stabilityin vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in β-lactamase evolution and a common mechanism in protein evolution.
10
Kapoor, Ritika R., Sarah E. Flanagan, Piers Fulton, Anupam Chakrapani, Bernadette Chadefaux, Tawfeg Ben-Omran, Indraneel Banerjee, Julian P. Shield, Sian Ellard, and Khalid Hussain. "Hyperinsulinism–hyperammonaemia syndrome: novel mutations in the GLUD1 gene and genotype–phenotype correlations." European Journal of Endocrinology 161, no. 5 (November 2009): 731–35. http://dx.doi.org/10.1530/eje-09-0615.
Full textAbstract:
BackgroundActivating mutations in the GLUD1 gene (which encodes for the intra-mitochondrial enzyme glutamate dehydrogenase, GDH) cause the hyperinsulinism–hyperammonaemia (HI/HA) syndrome. Patients present with HA and leucine-sensitive hypoglycaemia. GDH is regulated by another intra-mitochondrial enzyme sirtuin 4 (SIRT4). Sirt4 knockout mice demonstrate activation of GDH with increased amino acid-stimulated insulin secretion.ObjectivesTo study the genotype–phenotype correlations in patients with GLUD1 mutations. To report the phenotype and functional analysis of a novel mutation (P436L) in the GLUD1 gene associated with the absence of HA.Patients and methodsTwenty patients with HI from 16 families had mutational analysis of the GLUD1 gene in view of HA (n=19) or leucine sensitivity (n=1). Patients negative for a GLUD1 mutation had sequence analysis of the SIRT4 gene. Functional analysis of the novel P436L GLUD1 mutation was performed.ResultsHeterozygous missense mutations were detected in 15 patients with HI/HA, 2 of which are novel (N410D and D451V). In addition, a patient with a normal serum ammonia concentration (21 μmol/l) was heterozygous for a novel missense mutation P436L. Functional analysis of this mutation confirms that it is associated with a loss of GTP inhibition. Seizure disorder was common (43%) in our cohort of patients with a GLUD1 mutation. No mutations in the SIRT4 gene were identified.ConclusionPatients with HI due to mutations in the GLUD1 gene may have normal serum ammonia concentrations. Hence, GLUD1 mutational analysis may be indicated in patients with leucine sensitivity; even in the absence of HA. A high frequency of epilepsy (43%) was observed in our patients with GLUD1 mutations.
11
Demina, Anna, Kottayil I. Varughese, José Barbot, Linda Forman, and Ernest Beutler. "Six Previously Undescribed Pyruvate Kinase Mutations Causing Enzyme Deficiency." Blood 92, no. 2 (July 15, 1998): 647–52. http://dx.doi.org/10.1182/blood.v92.2.647.
Full textAbstract:
Abstract Erythrocyte pyruvate kinase deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia. We present 6 previously undescribed mutations of the PKLR gene associated with enzyme deficiency located at cDNA nt 476 G→T (159Gly→Val), 884 C→T (295Ala→Val), 943 G→A (315Glu→Lys), 1022 G→A (341Gly→Asp), 1511 G→T (504Arg→Leu), and 1528 C→T (510Arg→Ter). Two of these mutations are near the substrate binding site: the 315Glu→Lys (943A) mutation may be involved in Mg2+ binding and159Gly→Val (476T) mutation has a possible effect on ADP binding. Four of six mutations produce deduced changes in the shape of the molecule. Two of these mutations,504Arg→Leu (1511T) and510Arg→Ter (1528T), are located at the interface of domains A and C. One of them (510Arg→Ter) is a deletion of the C-terminal residues affecting the integrity of the protein. The 504Arg→Leu mutation eliminates a stabilizing interaction between domains A and C. Changes in amino acid 341(nt 1022) from Gly to Asp cause local perturbations. The mutation295Ala→Val (884T) might affect the way pyruvate kinase interacts with other molecules. We review previously described mutations and conclude that there is not yet sufficient data to allow us to draw conclusions regarding genotype/phenotype relationship.
12
Demina, Anna, Kottayil I. Varughese, José Barbot, Linda Forman, and Ernest Beutler. "Six Previously Undescribed Pyruvate Kinase Mutations Causing Enzyme Deficiency." Blood 92, no. 2 (July 15, 1998): 647–52. http://dx.doi.org/10.1182/blood.v92.2.647.414k13_647_652.
Full textAbstract:
Erythrocyte pyruvate kinase deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia. We present 6 previously undescribed mutations of the PKLR gene associated with enzyme deficiency located at cDNA nt 476 G→T (159Gly→Val), 884 C→T (295Ala→Val), 943 G→A (315Glu→Lys), 1022 G→A (341Gly→Asp), 1511 G→T (504Arg→Leu), and 1528 C→T (510Arg→Ter). Two of these mutations are near the substrate binding site: the 315Glu→Lys (943A) mutation may be involved in Mg2+ binding and159Gly→Val (476T) mutation has a possible effect on ADP binding. Four of six mutations produce deduced changes in the shape of the molecule. Two of these mutations,504Arg→Leu (1511T) and510Arg→Ter (1528T), are located at the interface of domains A and C. One of them (510Arg→Ter) is a deletion of the C-terminal residues affecting the integrity of the protein. The 504Arg→Leu mutation eliminates a stabilizing interaction between domains A and C. Changes in amino acid 341(nt 1022) from Gly to Asp cause local perturbations. The mutation295Ala→Val (884T) might affect the way pyruvate kinase interacts with other molecules. We review previously described mutations and conclude that there is not yet sufficient data to allow us to draw conclusions regarding genotype/phenotype relationship.
13
Kitanaka, Sachiko, Akiko Murayama, Toshiyuki Sakaki, Kuniyo Inouye, Yoshiki Seino, Seiji Fukumoto, Masaaki Shima, et al. "No Enzyme Activity of 25-Hydroxyvitamin D3 1α-Hydroxylase Gene Product in Pseudovitamin D Deficiency Rickets, Including That with Mild Clinical Manifestation." Journal of Clinical Endocrinology & Metabolism 84, no. 11 (November 1, 1999): 4111–17. http://dx.doi.org/10.1210/jcem.84.11.6131.
Full textAbstract:
Pseudovitamin D deficiency rickets (PDDR) is an autosomal recessive disorder caused by defect in the activation of vitamin D. We recently isolated 25-hydroxyvitamin D3 1α-hydroxylase gene and identified four homozygous inactivating missense mutations in this gene by analysis of four typical cases of PDDR. This disease shows some phenotypic variation, and it has been suspected that patients with mild phenotypes have mutations that do not totally abolish the enzyme activity. To investigate the molecular defects associated with the phenotypic variation, we analyzed six additional unrelated PDDR patients: one with mild and five with typical clinical manifestation. By sequence analysis, all six patients were proven to have mutations in both alleles. The mutations varied, and we identified four novel missense mutations, a nonsense mutation, and a splicing mutation for the first time. The patient with mild clinical symptoms was compound heterozygous for T321R and a splicing mutation. The splice site mutation caused intron retention. Enzyme activity of the T321R mutant was analyzed by overexpressing the mutant 1α-hydroxylase in Escherichia coli cells to detect the subtle residual enzyme activity. No residual enzyme activity was detected in T321R mutant or in the other mutants. These results indicate that all of the patients, including those of mild phenotype, are caused by 1α-hydroxylase gene mutations that totally abolish the enzyme activity.
14
Tharun, Sundaresan, and Roy Parker. "Analysis of Mutations in the Yeast mRNA Decapping Enzyme." Genetics 151, no. 4 (April 1, 1999): 1273–85. http://dx.doi.org/10.1093/genetics/151.4.1273.
Full textAbstract:
Abstract A major mechanism of mRNA decay in yeast is initiated by deadenylation, followed by mRNA decapping, which exposes the transcript to 5′ to 3′ exonucleolytic degradation. The decapping enzyme that removes the 5′ cap structure is encoded by the DCP1 gene. To understand the function of the decapping enzyme, we used alanine scanning mutagenesis to create 31 mutant versions of the enzyme, and we examined the effects of the mutations both in vivo and in vitro. Two types of mutations that affected mRNA decapping in vivo were identified, including a temperature-sensitive allele. First, two mutants produced decapping enzymes that were defective for decapping in vitro, suggesting that these mutated residues are required for enzymatic activity. In contrast, several mutants that moderately affected mRNA decapping in vivo yielded decapping enzymes that had at least the same specific activity as the wild-type enzyme in vitro. Combination of alleles within this group yielded decapping enzymes that showed a strong loss of function in vivo, but that still produced fully active enzymes in vitro. This suggested that interactions of the decapping enzyme with other factors may be required for efficient decapping in vivo, and that these particular mutations may be disrupting such interactions. Interestingly, partial loss of decapping activity in vivo led to a defect in normal deadenylation-dependent decapping, but it did not affect the rapid deadenylation-independent decapping triggered by early nonsense codons. This observation suggested that these two types of mRNA decapping differ in their requirements for the decapping enzyme.
15
NJÅLSSON, Runa, Katarina CARLSSON, Birgit OLIN, Birgit CARLSSON, Lel WHITBREAD, Galina POLEKHINA, Michael W. PARKER, et al. "Kinetic properties of missense mutations in patients with glutathione synthetase deficiency." Biochemical Journal 349, no. 1 (June 26, 2000): 275–79. http://dx.doi.org/10.1042/bj3490275.
Full textAbstract:
Patients with hereditary glutathione synthetase (GS) (EC 6.3.2.3) deficiency present with variable clinical pictures, presumably related to the nature of the mutations involved. In order to elucidate the relationship between genotype, enzyme function and clinical phenotype, we have characterized enzyme kinetic parameters of missense mutations R125C, R267W, R330C and G464V from patients with GS deficiency. One of the mutations predominantly affected the Km value, with decreased affinity for glycine, two mutations influenced both Km and Vmax values, and one mutation reduced the stability of the enzyme. This characterization agrees well with predictions based on the recently reported crystal structure of human GS. Thus our data indicate that different mutations can affect the catalytic capacity of GS by decreasing substrate affinity, maximal velocity or enzyme stability.
16
Boyer, Paul L., and Stephen H. Hughes. "Effects of Amino Acid Substitutions at Position 115 on the Fidelity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase." Journal of Virology 74, no. 14 (July 15, 2000): 6494–500. http://dx.doi.org/10.1128/jvi.74.14.6494-6500.2000.
Full textAbstract:
ABSTRACT We compared the fidelity of wild-type human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and two RT mutants, Y115F and Y115V. Although neither mutation had a large effect on the overall fidelity of the enzyme, both mutations altered the spectrum of mutations and the precise nature of the mutational hot spots. The effects of Y115V were greater than those of Y115F. When we compared the behavior of the wild-type enzyme with published data, we found that, in contrast to what has been published, misalignment/slippage could account for only a small fraction of the mutations we observed. We also found that a preponderance of the mutations (both transitions and transversions) resulted in the insertion of an A. Because we were measuring DNA-dependent DNA synthesis (plus-strand synthesis), this bias could contribute to the A-rich nature of the HIV-1 genome.
17
Petrović, Dušan, Valeria A. Risso, Shina Caroline Lynn Kamerlin, and Jose M. Sanchez-Ruiz. "Conformational dynamics and enzyme evolution." Journal of The Royal Society Interface 15, no. 144 (July 2018): 20180330. http://dx.doi.org/10.1098/rsif.2018.0330.
Full textAbstract:
Enzymes are dynamic entities, and their dynamic properties are clearly linked to their biological function. It follows that dynamics ought to play an essential role in enzyme evolution. Indeed, a link between conformational diversity and the emergence of new enzyme functionalities has been recognized for many years. However, it is only recently that state-of-the-art computational and experimental approaches are revealing the crucial molecular details of this link. Specifically, evolutionary trajectories leading to functional optimization for a given host environment or to the emergence of a new function typically involve enriching catalytically competent conformations and/or the freezing out of non-competent conformations of an enzyme. In some cases, these evolutionary changes are achieved through distant mutations that shift the protein ensemble towards productive conformations. Multifunctional intermediates in evolutionary trajectories are probably multi-conformational, i.e. able to switch between different overall conformations, each competent for a given function. Conformational diversity can assist the emergence of a completely new active site through a single mutation by facilitating transition-state binding. We propose that this mechanism may have played a role in the emergence of enzymes at the primordial, progenote stage, where it was plausibly promoted by high environmental temperatures and the possibility of additional phenotypic mutations.
18
Favor, Jack. "Risk estimation based on germ-cell mutations in animals." Genome 31, no. 2 (January 15, 1989): 844–52. http://dx.doi.org/10.1139/g89-149.
Full textAbstract:
The set of mouse germ cell mutation rate results following spermatogonial exposure to high dose rate irradiation have been presented as the most relevant experimental results upon which to extrapolate the expected genetic risk of offspring of the survivors of the Hiroshima and Nagasaki atomic bombings. Results include mutation rates to recessive specific-locus, dominant cataract, protein-charge, and enzyme-activity alleles. The mutability as determined by the various genetic end points differed: the mutation rates to recessive specific-locus alleles and enzyme-activity alleles were similar and greater than the mutation rates to dominant cataract and protein-charge alleles. It is argued that the type of mutation event scored by a particular test will determine the mutability of the genetic end point screened. When the loss of functional gene product can be scored in a particular mutation test, as in the recessive specific-locus and enzyme-activity tests, a wide spectrum of DNA alterations may result in a loss and a higher mutation rate is observed. When an altered gene product is scored, as in the dominant cataract and protein-charge tests, a narrower spectrum of DNA alterations is screened and a lower mutation rate is observed. The radiation doubling dose, defined as the dose that induces as many mutations as occur spontaneously per generation, was shown to be four times higher in the dominant cataract test than the specific-locus test. These results indicate that to extrapolate to genetic risks in humans using the doubling-dose method, the extrapolation must be based on experimental mutation rate results for the same genetic end point. Alternatively, the extrapolation could employ the direct-approach procedures. Finally, a direct comparison of the irradiation-induced mutation rate to enzyme-activity alleles in mouse and man indicates no species differences.Key words: ethylnitrosourea, irradiation, mammalian mutagenesis, mouse, dominant cataract mutations, specific-locus mutations, protein-charge mutations, enzyme-activity mutations, doubling dose, human genetic risk.
19
Mortimer, Sarah E., and Lizbeth Hedstrom. "Autosomal dominant retinitis pigmentosa mutations in inosine 5′-monophosphate dehydrogenase type I disrupt nucleic acid binding." Biochemical Journal 390, no. 1 (August 9, 2005): 41–47. http://dx.doi.org/10.1042/bj20042051.
Full textAbstract:
Two mutations of IMPDH1 (inosine 5′-monophosphate dehydrogenase type I), R224P and D226N, have recently been found to cause adRP (autosomal dominant retinitis pigmentosa). IMPDH1 catalyses the rate-limiting step in guanine nucleotide biosynthesis and also binds single-stranded nucleic acids. In the present paper, we report the biochemical characterization of the adRP-linked mutations, R224P and D226N, and a potentially pathogenic mutation, V268I. The adRP-linked mutations have no effect on enzyme activity, protein stability or protein aggregation. These results suggest strongly that the mutations do not affect enzyme activity in vivo and thus do not perturb the guanine nucleotide pool. The R224P mutation changes the distribution of enzyme between the nucleus and cytoplasm. This effect was not observed with the D226N mutation, so the relevance of this observation to disease is unclear. In contrast, both mutations decrease the affinity of nucleic acid binding and both fail to co-immunoprecipitate RNA. These observations suggest that nucleic acid binding provides a functional assay for adRP pathogenicity. The putative adRP-linked mutation V268I also disrupts nucleic acid binding, which suggests that this mutation is indeed pathogenic.
20
Klesmith, Justin R., and Benjamin J. Hackel. "Improved mutant function prediction via PACT: Protein Analysis and Classifier Toolkit." Bioinformatics 35, no. 16 (December 24, 2018): 2707–12. http://dx.doi.org/10.1093/bioinformatics/bty1042.
Full textAbstract:
Abstract Motivation Deep mutational scanning experiments have enabled the measurement of the sequence-function relationship for thousands of mutations in a single experiment. The Protein Analysis and Classifier Toolkit (PACT) is a Python software package that marries the fitness metric of a given mutation within these experiments to sequence and structural features enabling downstream analyses. PACT enables the easy development of user sharable protocols for custom deep mutational scanning experiments as all code is modular and reusable between protocols. Protocols for mutational libraries with single or multiple mutations are included. To exemplify its utility, PACT assessed two deep mutational scanning datasets that measured the tradeoff of enzyme activity and enzyme stability. Results PACT efficiently evaluated classifiers that predict protein mutant function tested on deep mutational scanning screens. We found that the classifiers with the lowest false positive and highest true positive rate assesses sequence homology, contact number and if mutation involves proline. Availability and implementation PACT and the processed datasets are distributed freely under the terms of the GPL-3 license. The source code is available at GitHub (https://github.com/JKlesmith/PACT). Supplementary information Supplementary data are available at Bioinformatics online.
21
Dothie, J. M., J. R. Giglio, C. B. Moore, S. S. Taylor, and B. S. Hartley. "Ribitol dehydrogenase of Klebsiella aerogenes. Sequence and properties of wild-type and mutant strains." Biochemical Journal 230, no. 3 (September 15, 1985): 569–78. http://dx.doi.org/10.1042/bj2300569.
Full textAbstract:
Evidence is presented for the sequence of 249 amino acids in ribitol dehydrogenase-A from Klebsiella aerogenes. Continuous culture on xylitol yields strains that superproduce ‘wild-type’ enzyme but mutations appear to have arisen in this process. Other strains selected by such continuous culture produce enzymes with increased specific activity for xylitol but without loss of ribitol activity. One such enzyme, ribitol dehydrogenase-D, has Pro-196 for Gly-196. Another, ribitol dehydrogenase-B, has a different mutation.
22
Vouillot, Léna, Aurore Thélie, and Nicolas Pollet. "Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases." G3 Genes|Genomes|Genetics 5, no. 3 (March 1, 2015): 407–15. http://dx.doi.org/10.1534/g3.114.015834.
Full textAbstract:
Abstract Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.
23
Cong, P., and S. Shuman. "Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA." Molecular and Cellular Biology 15, no. 11 (November 1995): 6222–31. http://dx.doi.org/10.1128/mcb.15.11.6222.
Full textAbstract:
Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7) methyltransferase, and transcription termination factor activities. The protein is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The capping reaction entails transfer of GMP from GTP to the 5'-diphosphate end of mRNA via a covalent enzyme-(lysyl-GMP) intermediate. The active site is situated at Lys-260 of the D1 subunit within a sequence element, KxDG (motif I), that is conserved in the capping enzymes from yeasts and other DNA viruses and at the active sites of covalent adenylylation of RNA and DNA ligases. Four additional sequence motifs (II to V) are conserved in the same order and with similar spacing among the capping enzymes and several ATP-dependent ligases. The relevance of these common sequence elements to the RNA capping reaction was addressed by mutational analysis of the vaccinia virus D1 protein. Nine alanine substitution mutations were targeted to motifs II to V. Histidine-tagged versions of the mutated D1 polypeptide were coexpressed in bacteria with the D12 subunit, and the His-tagged heterodimers were purified by Ni affinity and phosphocellulose chromatography steps. Whereas each of the mutated enzymes retained triphosphatase, methyltransferase, and termination factor activities, six of nine mutant enzymes were defective in some aspect of transguanylylation. Individual mutations in motifs III, IV, and V had distinctive effects on the affinity of enzyme for GTP, the rate of covalent catalysis (EpG formation), or the transfer of GMP from enzyme to RNA. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a conserved structural basis for covalent nucleotidyl transfer.
24
Chelstowska, A., T. Zoladek, J. Garey, J. Kushner, J. Rytka, and R. Labbe-Bois. "Identification of amino acid changes affecting yeast uroporphyrinogen decarboxylase activity by sequence analysis of hem12 mutant alleles." Biochemical Journal 288, no. 3 (December 15, 1992): 753–57. http://dx.doi.org/10.1042/bj2880753.
Full textAbstract:
The molecular basis of the uroporphyrinogen decarboxylase defect in eleven yeast ‘uroporphyric’ mutants was investigated. Uroporphyrinogen decarboxylase, an enzyme of the haem-biosynthetic pathway, catalyses the decarboxylation of uroporphyrinogen to coproporphyrinogen and is encoded by the HEM12 gene in the yeast Saccharomyces cerevisiae. The mutations were identified by sequencing the mutant hem12 alleles amplified in vitro from genomic DNA extracted from the mutant strains. Four mutations leading to the absence of enzyme protein were found: one mutation caused the substitution of the translation initiator Met to Ile, a two-base deletion created a frameshift at codon 247 and two nonsense mutations were found at codons 50 and 263. Four different point mutations were identified in seven ‘leaky’ mutants with residual modified uroporphyrinogen decarboxylase activity; each of three mutations was found in two independently isolated mutants. The nucleotide transitions resulted in the amino acid substitutions Ser-59 to Phe, Thr-62 to Ile, Leu-107 to Ser, or Ser-215 to Asn, all located in or near highly conserved regions. The results suggest that there is a single active centre in uroporphyrinogen decarboxylase, the geometry of which is affected in the mutant enzymes.
25
Jin, Da-Yun, Jian-Ke Tie, and Darrel W. Stafford. "Mutational Analysis of VKOR." Blood 108, no. 11 (November 16, 2006): 1628. http://dx.doi.org/10.1182/blood.v108.11.1628.1628.
Full textAbstract:
Abstract More than 21 million prescriptions for warfarin are written yearly in the US for Vitamin K epoxide reductase (VKOR), the target of warfarin The vitamin K epoxide reductase, VKOR, apparently uses cysteines, 132 and 135 as active sites. In addition to cysteines 132 and 135, cysteines 43 and 51 are conserved throughout evolution. Rost et al. have mutated each of the cysteines in VKOR and found that, in whole cell lysates, mutations in of C43 result in less than 20% of wild-type activity while mutation of C53 eliminates activity. We have repeated these experiments and mutated all of the cysteine residues in VKOR. Our results in microsomes are similar to the results of Rost et. al. (2005) Thromb. Haemost. 94, 780–786. However, when the mutated enzymes are purified we find that the activity of C43 has 30% residual activity while C51 has 60% residual activity. Mutation of both residues in the same molecule results in an enzyme that is similar to the C51 mutation. In addition, we find that a portion of purified VKOR has a disulfide bond between residues 43 and 51. This suggested that we might be able to remove the loop between C43 and C51 and retain activity. Indeed, the mutated enzyme with this loop removed also has substantial activity. Warfarin inhibition studies suggest that these mutations do not materially affect warfarin sensitivity. Our results stress the importance of utilizing purified enzyme for interpreting the results of mutations in VKOR.
26
Kaneko, Mika, Shoko Nishihara, Naoko Shinya, Takashi Kudo, Hiroko Iwasaki, Taiko Seno, Yasuto Okubo, and Hisashi Narimatsu. "Wide Variety of Point Mutations in the H Gene of Bombay and Para-Bombay Individuals That Inactivate H Enzyme." Blood 90, no. 2 (July 15, 1997): 839–49. http://dx.doi.org/10.1182/blood.v90.2.839.
Full textAbstract:
Abstract The H genes, encoding an α1,2fucosyltransferase, which defines blood groups with the H structure, of four Bombay and 13 para-Bombay Japanese individuals were analyzed for mutations. Four Bombay individuals were homologous for the same null H allele, which is inactivated by a single nonsense mutation at position 695 from G to A (G695A), resulting in termination of H gene translation. The allele inactivated by the G695A was designated h1. The other 13 para-Bombay individuals possessed a trace amount of H antigens on erythrocytes regardless of their secretor status. Sequence analysis of their H genes showed four additional inactivated H gene alleles, h2, h3, h4, and h5. The h2 allele possesed a single base deletion at position 990 G (990-del). The h3 and h4 alleles possessed a single missense mutation, T721C, which changes Tyr 241 to His, and G442T, which changes Asp148 to Tyr, respectively. The h5 allele possessed two missense mutations, T460C (Tyr154 to His) and G1042A (Glu348 to Lys). The h2, h3, h4, and h5 enzymes directed by these alleles were not fully inactivated by the deletion and the missense mutations expressing some residual enzyme activity resulting in synthesis of H antigen on erythrocytes. Thirteen para-Bombay individuals whose erythrocytes retained a trace amount of H antigen were determined to be heterozygous or homozygous for at least one of h2, h3, h4, or h5 alleles. This clarified that the levels (null to trace amount) of H antigen expression on erythrocytes of Bombay and para-Bombay individuals are determined solely by H enzyme activity. These mutations found in the Japanese H alleles differ from a nonsense mutation found in the Indonesian population. To determine the roles of the H, Se, and Le genes in the expression of H antigen in secretions and Lewis blood group antigen on erythrocytes, the Lewis and secretor genes were also examined in these Bombay and para-Bombay individuals. The Lewis blood group phenotype, Le(α- b+), was determined by the combinatorial activity of two fucosyltransferases, the Lewis enzyme and the secretor enzyme, and the secretor status was solely determined by the secretor enzyme activity, not by H enzyme activity. Bombay individuals were confirmed to be homozygous for the inactivated H and Se genes. As expected from the very low frequency of Bombay and para-Bombay individuals in the population, ie, approximately one in two or 300,000, the H gene mutations were found to be very variable, unlike the cases of the point mutations in the other glycosyltransferase genes; the ABO genes, the Lewis gene, and the secretor gene.
27
Kaneko, Mika, Shoko Nishihara, Naoko Shinya, Takashi Kudo, Hiroko Iwasaki, Taiko Seno, Yasuto Okubo, and Hisashi Narimatsu. "Wide Variety of Point Mutations in the H Gene of Bombay and Para-Bombay Individuals That Inactivate H Enzyme." Blood 90, no. 2 (July 15, 1997): 839–49. http://dx.doi.org/10.1182/blood.v90.2.839.839_839_849.
Full textAbstract:
The H genes, encoding an α1,2fucosyltransferase, which defines blood groups with the H structure, of four Bombay and 13 para-Bombay Japanese individuals were analyzed for mutations. Four Bombay individuals were homologous for the same null H allele, which is inactivated by a single nonsense mutation at position 695 from G to A (G695A), resulting in termination of H gene translation. The allele inactivated by the G695A was designated h1. The other 13 para-Bombay individuals possessed a trace amount of H antigens on erythrocytes regardless of their secretor status. Sequence analysis of their H genes showed four additional inactivated H gene alleles, h2, h3, h4, and h5. The h2 allele possesed a single base deletion at position 990 G (990-del). The h3 and h4 alleles possessed a single missense mutation, T721C, which changes Tyr 241 to His, and G442T, which changes Asp148 to Tyr, respectively. The h5 allele possessed two missense mutations, T460C (Tyr154 to His) and G1042A (Glu348 to Lys). The h2, h3, h4, and h5 enzymes directed by these alleles were not fully inactivated by the deletion and the missense mutations expressing some residual enzyme activity resulting in synthesis of H antigen on erythrocytes. Thirteen para-Bombay individuals whose erythrocytes retained a trace amount of H antigen were determined to be heterozygous or homozygous for at least one of h2, h3, h4, or h5 alleles. This clarified that the levels (null to trace amount) of H antigen expression on erythrocytes of Bombay and para-Bombay individuals are determined solely by H enzyme activity. These mutations found in the Japanese H alleles differ from a nonsense mutation found in the Indonesian population. To determine the roles of the H, Se, and Le genes in the expression of H antigen in secretions and Lewis blood group antigen on erythrocytes, the Lewis and secretor genes were also examined in these Bombay and para-Bombay individuals. The Lewis blood group phenotype, Le(α- b+), was determined by the combinatorial activity of two fucosyltransferases, the Lewis enzyme and the secretor enzyme, and the secretor status was solely determined by the secretor enzyme activity, not by H enzyme activity. Bombay individuals were confirmed to be homozygous for the inactivated H and Se genes. As expected from the very low frequency of Bombay and para-Bombay individuals in the population, ie, approximately one in two or 300,000, the H gene mutations were found to be very variable, unlike the cases of the point mutations in the other glycosyltransferase genes; the ABO genes, the Lewis gene, and the secretor gene.
28
Kim, Young-Wan, Ji-Hye Choi, Jung-Wan Kim, Cheonseok Park, Jung-Woo Kim, Hyunju Cha, Soo-Bok Lee, Byoung-Ha Oh, Tae-Wha Moon, and Kwan-Hwa Park. "Directed Evolution of Thermus Maltogenic Amylase toward Enhanced Thermal Resistance." Applied and Environmental Microbiology 69, no. 8 (August 2003): 4866–74. http://dx.doi.org/10.1128/aem.69.8.4866-4874.2003.
Full textAbstract:
ABSTRACT The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.
29
Matange, Nishad, Swapnil Bodkhe, Maitri Patel, and Pooja Shah. "Trade-offs with stability modulate innate and mutationally acquired drug resistance in bacterial dihydrofolate reductase enzymes." Biochemical Journal 475, no. 12 (June 29, 2018): 2107–25. http://dx.doi.org/10.1042/bcj20180249.
Full textAbstract:
Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In the present study, we show that trimethoprim (TMP)-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harboring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild-type, making them prone to aggregation and proteolysis. This destabilization is associated with a lower expression level, resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis, we show that perturbation of critical stabilizing hydrophobic interactions in wild-type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial dihydrofolate reductases (DHFRs). Mutational and computational analyses in EcDHFR and in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues modulates TMP resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to TMP. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme.
30
Romero, Philip A., Tuan M. Tran, and Adam R. Abate. "Dissecting enzyme function with microfluidic-based deep mutational scanning." Proceedings of the National Academy of Sciences 112, no. 23 (May 26, 2015): 7159–64. http://dx.doi.org/10.1073/pnas.1422285112.
Full textAbstract:
Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme’s sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence–function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.
31
Gokgoz, Zafer, Veysel Sabri Hancer, and Reyhan Diz-Kucukkaya. "Two Novel Mutations (p.I454T and p.Y472X) and a Homozygous p.A109T Mutation Associated with Factor XI Deficiency in a Turkish Family." Blood 124, no. 21 (December 6, 2014): 5054. http://dx.doi.org/10.1182/blood.v124.21.5054.5054.
Full textAbstract:
Abstract Introduction: Factor XI (FXI) is a homodimeric serine protease enzyme, which is produced in the liver and circulates in the plasma complexed with high molecular weight kininogen. Approximately 1% of FXI is presented in platelets. After the activation of the extrinsic pathway of coagulation, thrombin activates FXI, and this reaction has an important role in the amplification of coagulation reaction. Congenital FXI deficiency is characterized by decreased levels or activity of FXI in the plasma, and may cause an inherited bleeding disorder. Congenital FXI deficiency is very rare (less than 1/100.000) in the general population. The factor XI gene is located on the long arm of chromosome 4 (4q34-35), consists of 15 exons and 14 introns, and encodes a 607 amino acid protein. FXIa has two chains: the light chain contains the catalytic site whereas the heavy chain contains the four tandem repeat sequences (Apple domains, A1 to A4). Mutations of the FXI gene (F11 gene) leading to FXI deficiency can occur in the Apple domains or the catalytic site. One of the most common mutations is a missense mutation in the Apple 4 domain, which is important for FXI dimerization, resulting in low serum levels of FXI. Some mutations can cause a moderate to severe FXI deficiency by producing aberrant proteins. Here we present a consanguineous family with FXI deficiency. Patients and methods: In this study a family with FXI deficiency was evaluated. The index patient was a 10 years old boy with bleeding diathesis (excessive bleeding after tooth extraction). His aPTT level was 84,3 seconds (N: 32-39 seconds), FXI activity was 0%, and he was diagnosed with FXI deficiency in 2010. His parents were related. The father had a mild bleeding tendency with prolonged aPTT (48.2 seconds). FXI activity was found to be 4%. The mother and second child had no bleeding history with mildly decreased FXI activities (40% and 60%, respectively). We performed a mutational analysis for whole family, including his grandparents. Genomic DNA was extracted from whole blood. All exons and approximately 30bp exon-intron boundaries of F11 gene were amplified using sets of designed primers. PCR reactions were performed using 2X PCR master mix (HibriGen Biotech R&D, Istanbul, Turkey), primers and 25-30 ng genomic DNA. Amplified fragments were sequenced. Results: We obtained the results shown in Figure 1. Figure 1: The pedigree of the patient. Figure 1:. The pedigree of the patient. The patient and his father had a A109T homozygous mutation for F11; the index patient also had heterozygous I454T and Y472X mutations. The presence of a homozygous A109T mutation in father and index patient caused severe FXI deficiency. The mother and second child had heterozygous I454T and Y472X mutations. As shown in the pedigree, I454T and Y472X compound heterozygosity moderately decreases the activity of FXI. Conclusions: Approximately 220 F11 mutations have been reported to date, affecting both the catalytic and Apple domains. In this family, we found two novel mutations, I454T and Y472X, associated with a homozygous A109T mutation. This is the first case reported with a homozygous A109T mutation in the literature. Previously Guella et al. reported a heterozygous A109T mutation in an Italian family with FXI deficiency (Thromb Haemost, 2008). They showed that exon-skipping had occured due to a heterozygous A109T mutation. They explained that the unchanged enzyme activity was due to a non-sense mediated RNA decay mechanism. Our cases confirmed their results, such that a heterozygous A109T mutation did not affect enzyme activity; the enzyme activity of a person who has two heterozygous mutations (Y472X and I454T) is the same as who has three heterozygous mutations (A109T, Y472X and I454T). However, when the A109T was homozygous like in our patient and his father, the enzyme activity decreased by approximately 96% as shown in the pedigree. Another interesting point was the presence of a homozygous A109T mutation in the patient while the mother had no A109T mutation. This can be explained by somatic recombination as seen in variable diversity junction (VDJ) recombination in the immune system. Further expression studies evaluating the effects of these mutations will improve our understanding of the functional and structural features of the FXI enzyme. Disclosures No relevant conflicts of interest to declare.
32
Dekker, Jan, Michel H. M. Eppink, Rob van Zwieten, Thea de Rijk, Angel F. Remacha, Lap Kay Law, Albert M. Li, Kam Lau Cheung, Willem J. H. van Berkel, and Dirk Roos. "Seven new mutations in the nicotinamide adenine dinucleotide reduced–cytochrome b5 reductase gene leading to methemoglobinemia type I." Blood 97, no. 4 (February 15, 2001): 1106–14. http://dx.doi.org/10.1182/blood.v97.4.1106.
Full textAbstract:
Abstract Cytochrome b5 reductase (b5R) deficiency manifests itself in 2 distinct ways. In methemoglobinemia type I, the patients only suffer from cyanosis, whereas in type II, the patients suffer in addition from severe mental retardation and neurologic impairment. Biochemical data indicate that this may be due to a difference in mutations, causing enzyme instability in type I and complete enzyme deficiency or enzyme inactivation in type II. We have investigated 7 families with methemoglobulinemia type I and found 7 novel mutations in the b5R gene. Six of these mutations predicted amino acid substitutions at sites not involved in reduced nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD) binding, as deduced from a 3-dimensional model of human b5R. This model was constructed from comparison with the known 3-dimensional structure of pig b5R. The seventh mutation was a splice site mutation leading to skipping of exon 5 in messenger RNA, present in heterozygous form in a patient together with a missense mutation on the other allele. Eight other amino acid substitutions, previously described to cause methemoglobinemia type I, were also situated in nonessential regions of the enzyme. In contrast, 2 other substitutions, known to cause the type II form of the disease, were found to directly affect the consensus FAD-binding site or indirectly influence NADH binding. Thus, these data support the idea that enzyme inactivation is a cause of the type II disease, whereas enzyme instability may lead to the type I form.
33
Jindal, Garima, Katerina Slanska, Veselin Kolev, Jiri Damborsky, Zbynek Prokop, and Arieh Warshel. "Exploring the challenges of computational enzyme design by rebuilding the active site of a dehalogenase." Proceedings of the National Academy of Sciences 116, no. 2 (December 26, 2018): 389–94. http://dx.doi.org/10.1073/pnas.1804979115.
Full textAbstract:
Rational enzyme design presents a major challenge that has not been overcome by computational approaches. One of the key challenges is the difficulty in assessing the magnitude of the maximum possible catalytic activity. In an attempt to overcome this challenge, we introduce a strategy that takes an active enzyme (assuming that its activity is close to the maximum possible activity), design mutations that reduce the catalytic activity, and then try to restore that catalysis by mutating other residues. Here we take as a test case the enzyme haloalkane dehalogenase (DhlA), with a 1,2-dichloroethane substrate. We start by demonstrating our ability to reproduce the results of single mutations. Next, we design mutations that reduce the enzyme activity and finally design double mutations that are aimed at restoring the activity. Using the computational predictions as a guide, we conduct an experimental study that confirms our prediction in one case and leads to inconclusive results in another case with 1,2-dichloroethane as substrate. Interestingly, one of our predicted double mutants catalyzes dehalogenation of 1,2-dibromoethane more efficiently than the wild-type enzyme.
34
Shoolingin-Jordan, P. M., P. Spencer, M. Sarwar, P. E. Erskine, K. M. Cheung, J. B. Cooper, and E. B. Norton. "5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism." Biochemical Society Transactions 30, no. 4 (August 1, 2002): 584–90. http://dx.doi.org/10.1042/bst0300584.
Full textAbstract:
5-Aminolaevulinic acid dehydratase catalyses the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. The studies described highlight the importance of a bivalent metal ion and two active-site lysine residues for the functioning of 5-aminolaevulinic acid dehydratase. Dehydratases fall into two main categories: zinc-dependent enzymes and magnesium-dependent enzymes. Mutations that introduced zinc-binding ligands into a magnesium-dependent enzyme conferred an absolute requirement for zinc. Mutagenesis of lysine residues 247 and 195 in the Escherichia coli enzyme lead to dramatic effects on enzyme activity, with lysine 247 being absolutely essential. Mutation of either lysine 247 or 195 to cysteine, and treatment of the mutant enzyme with 2-bromethylamine, resulted in the recovery of substantial enzyme activity. The effects of the site-directed alkylating inhibitor, 5-chlorolaevulinic acid, and 4,7-dioxosebacic acid, a putative intermediate analogue, were investigated by X-ray crystallography. These inhibitors reacted with both active-site lysine residues. The role of these two lysine residues in the enzyme mechanism is discussed.
35
Rogozin, Igor, Abiel Roche-Lima, Artem Lada, Frida Belinky, Ivan Sidorenko, Galina Glazko, Vladimir Babenko, David Cooper, and Youri Pavlov. "Nucleotide Weight Matrices Reveal Ubiquitous Mutational Footprints of AID/APOBEC Deaminases in Human Cancer Genomes." Cancers 11, no. 2 (February 12, 2019): 211. http://dx.doi.org/10.3390/cancers11020211.
Full textAbstract:
Cancer genomes accumulate nucleotide sequence variations that number in the tens of thousands per genome. A prominent fraction of these mutations is thought to arise as a consequence of the off-target activity of DNA/RNA editing cytosine deaminases. These enzymes, collectively called activation induced deaminase (AID)/APOBECs, deaminate cytosines located within defined DNA sequence contexts. The resulting changes of the original C:G pair in these contexts (mutational signatures) provide indirect evidence for the participation of specific cytosine deaminases in a given cancer type. The conventional method used for the analysis of mutable motifs is the consensus approach. Here, for the first time, we have adopted the frequently used weight matrix (sequence profile) approach for the analysis of mutagenesis and provide evidence for this method being a more precise descriptor of mutations than the sequence consensus approach. We confirm that while mutational footprints of APOBEC1, APOBEC3A, APOBEC3B, and APOBEC3G are prominent in many cancers, mutable motifs characteristic of the action of the humoral immune response somatic hypermutation enzyme, AID, are the most widespread feature of somatic mutation spectra attributable to deaminases in cancer genomes. Overall, the weight matrix approach reveals that somatic mutations are significantly associated with at least one AID/APOBEC mutable motif in all studied cancers.
36
Yen, Tina, Brian N. Nightingale, Jennifer C. Burns, David R. Sullivan, and Peter M. Stewart. "Butyrylcholinesterase (BCHE) Genotyping for Post-Succinylcholine Apnea in an Australian Population." Clinical Chemistry 49, no. 8 (August 1, 2003): 1297–308. http://dx.doi.org/10.1373/49.8.1297.
Full textAbstract:
Abstract Background: Measurement of plasma butyrylcholinesterase (BChE) activity and inhibitor-based phenotyping are standard methods for identifying patients who experience post-succinylcholine (SC) apnea attributable to inherited variants of the BChE enzyme. Our aim was to develop PCR-based assays for BCHE mutation detection and implement them for routine diagnostic use at a university teaching hospital. Methods: Between 1999 and 2002, we genotyped 65 patients referred after prolonged post-SC apnea. Five BCHE gene mutations were analyzed. Competitive oligo-priming (COP)-PCR was used for flu-1, flu-2, and K-variant and direct DNA sequencing analysis for dibucaine and sil-1 mutations. Additional DNA sequencing of BCHE coding regions was provided when the five-mutation screen was negative or mutation findings were inconsistent with enzyme activity. Results: Genotyping identified 52 patients with primary hypocholinesterasemia attributable to BCHE mutations, and in 44 individuals the abnormalities were detected by the five-mutation screen (detection rate, 85%). Additional sequencing studies revealed mutations in eight other patients, including five with novel mutations. The most common genotype abnormality was compound homozygous dibucaine and homozygous K-variant mutations. No simple homozygotes were found. Of the remaining 13 patients, 3 had normal BChE activity and gene, and 10 were diagnosed with hypocholinesterasemia unrelated to BCHE gene abnormalities. Conclusion: A five-mutation screen for investigation of post-SC apnea identified BCHE gene abnormalities for 80% of a referral population. Six new BCHE mutations were identified by sequencing studies of 16 additional patients.
37
Kraut, Daniel A., Paul A. Sigala, Timothy D. Fenn, and Daniel Herschlag. "Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole." Proceedings of the National Academy of Sciences 107, no. 5 (January 11, 2010): 1960–65. http://dx.doi.org/10.1073/pnas.0911168107.
Full textAbstract:
The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by 105-fold, far larger than the effects of hydrogen bond mutations in other enzymes. However, the less-conservative Tyr16Ser mutation, which also perturbs the Tyr16 hydrogen bond, results in a less-severe 102-fold rate reduction. To understand the paradoxical effects of these mutations and clarify the energetic importance of the Tyr16 hydrogen bond, we have determined the 1.6-Å resolution x-ray structure of the intermediate analogue, equilenin, bound to the Tyr16Ser mutant and measured the rate effects of mutating Tyr16 to Ser, Thr, Ala, and Gly. The nearly identical 200-fold rate reductions of these mutations, together with the 6.4-Å distance observed between the Ser16 hydroxyl and equilenin oxygens in the x-ray structure, strongly suggest that the more moderate rate effect of this mutant is not due to maintenance of a hydrogen bond from Ser at position 16. These results, additional spectroscopic observations, and prior structural studies suggest that the Tyr16Phe mutation results in unfavorable interactions with the dienolate intermediate beyond loss of a hydrogen bond, thereby exaggerating the apparent energetic benefit of the Tyr16 hydrogen bond relative to the solution reaction. These results underscore the complex energetics of hydrogen bonding interactions and site-directed mutagenesis experiments.
38
Alfonso, Pilar, Javier Gervas, Vanesa Andreu, Joaquin Navascues, Francisca Sanchez-Jimenez, Miguel Pocovi, Carmen Ortiz-Mellet, Jose M. Garcia-Fernandez, and Pilar Giraldo. "New Derivatives of L-Idonojirimycin Working As Pharmacological Chaperones for the Treatment of Gaucher Disease." Blood 120, no. 21 (November 16, 2012): 3270. http://dx.doi.org/10.1182/blood.v120.21.3270.3270.
Full textAbstract:
Abstract Abstract 3270 Introduction: Many of the mutations of the lysosomal acid b-glucosidase (b-glucocerebrosidase) associated with Gaucher disease (GD) translate into enzymes that retain partial catalytic activity in vitro but exhibit impaired cellular trafficking as a consequence of aberrant folding. Current investigational therapeutic strategies for include the development of ligands of the enzyme capable of promoting those conformational changes that are required for efficient folding, restoring trafficking. Although somewhat counter intuitive, competitive inhibitors of this b-glucocerebrosidase, at subinhibitory concentrations, can increase steady-state lysosomal levels of active enzyme through this rescuing mechanism, acting as “pharmacological chaperones”. At the massive lysosomal substrate concentration, the inhibitor would be replaced from the active site of the enzyme and the metabolic activity recovered. However, most of the pharmacological chaperones under study are iminosugars that behave as broad spectrum inhibitors, inhibiting simultaneously several glucosidases, which represents a serious inconvenient for clinical applications. An additional problem is that iminosugars and their derivatives are not active as pharmacological chaperones for glucocerebrosidase mutations located outside the domain containing the active site and are associated with neurological involvement, as the L444P mutation. Aim: The aim of this work is to present molecules with a high binding specificity towards b-glucocerebrosidase, with a high ratio of chaperone versus inhibitor activity and capable of producing an increased in the levels of mutant enzymes associated with Gaucher disease, including mutations located outside the catalytic domain. Methods: Different bicyclic derivatives of L-idonojirimycin were designed and chemically synthesized from D-glucose after in silico structural analysis and identification of the most favorable molecular features to interact with the active site of glucocerebrosidase. The chaperone potential of these compounds was evaluated at different concentrations in vitro using a cell model of GDcarrying the more frequent mutations in Gaucher disease, namely N370S and L444P. (P201230804). Results: The obtained results showed an increase in b-glucocerebrosidase activity at various chaperone concentrations, ranging from 1.96 to 4.98 folds for the L444P mutant and from 2.01 to 3.06 folds for the N370S mutation. Comments: The bicyclic derivatives of L-idonojirimycin could be considered as a therapeutic alternative for GD, mainly in patients with mutations located outside the active site of the enzyme and associated with neurologic involvement. Disclosures: Giraldo: Actelion: Membership on an entity's Board of Directors or advisory committees; Genzyme: Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding.
39
Maskell, Jeffrey P., Armine M. Sefton, and Lucinda M. C. Hall. "Multiple Mutations Modulate the Function of Dihydrofolate Reductase in Trimethoprim-ResistantStreptococcus pneumoniae." Antimicrobial Agents and Chemotherapy 45, no. 4 (April 1, 2001): 1104–8. http://dx.doi.org/10.1128/aac.45.4.1104-1108.2001.
Full textAbstract:
ABSTRACT Trimethoprim resistance in Streptococcus pneumoniaecan be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC50], 4.2 μM) than was the DHFR from strain CP1015 (IC50, 0.09 μM). However,K m values indicated a lower affinity for the enzyme's natural substrates (K m for dihydrofolate [DHF], 3.1 μM for CP1015 and 27.5 μM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, K m values for DHF were reduced (9.2 to 15.2 μM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC50 of up to 13.7 μM). It is postulated that carriage of the mutation M53-I—which, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR—is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates.
40
D’Argenio, David A., Mathew W. Vetting, Douglas H. Ohlendorf, and L. Nicholas Ornston. "Substitution, Insertion, Deletion, Suppression, and Altered Substrate Specificity in Functional Protocatechuate 3,4-Dioxygenases." Journal of Bacteriology 181, no. 20 (October 15, 1999): 6478–87. http://dx.doi.org/10.1128/jb.181.20.6478-6487.1999.
Full textAbstract:
ABSTRACT Protocatechuate 3,4-dioxygenase is a member of a family of bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic background, it is possible to select for Acinetobacterstrains containing spontaneous mutations blocking expression ofpcaH or -G, genes encoding the α and β subunits of protocatechuate 3,4-dioxygenase. The crystal structure of the Acinetobacter oxygenase has been determined, and this knowledge affords us the opportunity to understand how mutations alter function in the enzyme. An earlier investigation had shown that a large fraction of spontaneous mutations inactivatingAcinetobacter protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant selection was modified to isolate Acinetobacter strains in which mutations within pcaH or -G cause a heat-sensitive phenotype. These mutations affected residues distributed throughout the linear amino acid sequences of PcaH and PcaG and impaired the dioxygenase to various degrees. Four of 16 mutants had insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlighting areas of the protein where large structural changes can be tolerated. To further understand how protein structure influences function, we isolated strains in which the phenotypes of three different deletion mutations in pcaH or -G were suppressed either by a spontaneous mutation or by a PCR-generated random mutation introduced into theAcinetobacter chromosome by natural transformation. The latter procedure was also used to identify a single amino acid substitution in PcaG that conferred activity towards catechol sufficient for growth with benzoate in a strain in which catechol 1,2-dioxygenase was inactivated.
41
Lerner, Claude G., Stephan J. Kakavas, Christian Wagner, Richard T. Chang, Philip J. Merta, Xiaoan Ruan, Randy E. Metzger, and Bruce A. Beutel. "Novel Approach to Mapping of Resistance Mutations in Whole Genomes by Using Restriction Enzyme Modulation of Transformation Efficiency." Antimicrobial Agents and Chemotherapy 49, no. 7 (July 2005): 2767–77. http://dx.doi.org/10.1128/aac.49.7.2767-2777.2005.
Full textAbstract:
ABSTRACT Restriction enzyme modulation of transformation efficiencies (REMOTE) is a method that makes use of genome restriction maps and experimentally observed differences in transformation efficiencies of genomic DNA restriction digests to discover the location of mutations in genomes. The frequency with which digested genomic DNA from a resistant strain transforms a susceptible strain to resistance is primarily determined by the size of the fragment containing the resistance mutation and the distance of the mutation to the end of the fragment. The positions of restriction enzyme cleavage sites immediately flanking the resistance mutation define these parameters. The mapping procedure involves a process of elimination in which digests that transform with high frequency indicate that the restriction enzyme cleavage sites are relatively far away from the mutation, while digests that transform with low frequency indicate that the sites are close to the mutation. The transformation data are compared computationally to the genome restriction map to identify the regions that best fit the data. Transformations with PCR amplicons encompassing candidate regions identify the resistance locus and enable identification of the mutation. REMOTE was developed using Haemophilus influenzae strains with mutations in gyrA, gyrB, and rpsE that confer resistance to ciprofloxacin, novobiocin, and spectinomycin, respectively. We applied REMOTE to identify mutations that confer resistance to two novel antibacterial compounds. The resistance mutations were found in genes that can decrease the intracellular concentration of compounds: acrB, which encodes a subunit of the AcrAB-TolC efflux pump; and fadL, which encodes a long-chain fatty acid transporter.
42
Li, Yun, Kun Song, Jian Zhang, and Shaoyong Lu. "A Computational Method to Predict Effects of Residue Mutations on the Catalytic Efficiency of Hydrolases." Catalysts 11, no. 2 (February 22, 2021): 286. http://dx.doi.org/10.3390/catal11020286.
Full textAbstract:
With scientific and technological advances, growing research has focused on engineering enzymes that acquire enhanced efficiency and activity. Thereinto, computer-based enzyme modification makes up for the time-consuming and labor-intensive experimental methods and plays a significant role. In this study, for the first time, we collected and manually curated a data set for hydrolases mutation, including structural information of enzyme-substrate complexes, mutated sites and Kcat/Km obtained from vitro assay. We further constructed a classification model using the random forest algorithm to predict the effects of residue mutations on catalytic efficiency (increase or decrease) of hydrolases. This method has achieved impressive performance on a blind test set with the area under the receiver operating characteristic curve of 0.86 and the Matthews Correlation Coefficient of 0.659. Our results demonstrate that computational mutagenesis has an instructive effect on enzyme modification, which may expedite the design of engineering hydrolases.
43
Morera, Simone, Laurent Chiarelli, Stefano Rovida, Paola Bianchi, Elisa Fermo, Alessandro Galizzi, Alberto Zanella, and Giovanna Valentini. "Phosphoglycerate Kinase Deficiency: Characterization of the Wild-Type Enzyme and Three Pathological Variants Generated from C.140T>a, C.491A>T and C.959G>a Mutations." Blood 112, no. 11 (November 16, 2008): 2875. http://dx.doi.org/10.1182/blood.v112.11.2875.2875.
Full textAbstract:
Abstract Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phoshoryl-group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP forming 3-phosphoglycerate (3-PG) and ATP. PGK is a typical two-domain hinge-bending enzyme, with a highly conserved structure. The N-terminal domain binds 1,3-BPG/3-PG, whereas the C-terminal domain binds Mg-ADP/Mg-ATP.Humans have two PGK isozymes, PGK1 and PGK2, where PGK1 is an ubiquitous enzyme that is expressed in all somatic cells and PGK2 is a testis-specific enzyme. The PGK1 gene is located on the X-chromosome q-13.1, contains 11 exons and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in an enzyme deficiency that is for the most clinically characterized by mild-to severe hemolytic anemia and various defects in the central nervous system. To date, 19 different mutations with worldwide distribution have been reported. No correlation between the residual PGK activity and the severity of the clinical manifestations have been documented so far. To analyze the mutations at protein level and possibly to correlate the genotype to clinical phenotype, we started with the molecular characterization of the wild-type PGK1 enzyme and three mutants (I47N, D164 and S320N) obtained from E.coli as recombinant proteins. The corresponding mutations, i.e., c.140T>A, c.491A>T and c.959G>A, have been identified in patients with PGK deficiency and affected by severe hemolytic anemia and progressive mental retardation. The cDNA encoding the PGK1 was prepared starting from a blood sample of a healthy donor, with normal PGK1 activity. Site-directed mutagenesis was used to introduce the desired mutations into the PGK1 cDNA. The wild type enzyme was expressed to its maximum level (about 80–100 mg of enzyme per liter of culture) after 5 hours of induction with 0.5 mM IPTG at 37 °C. For mutant enzymes the induction temperature was lowered to 25°C. All recombinant enzymes were purified to homogeneity after a single chromatographic step on DEAE Sepharose column. The wild-type enzyme was crystallized in both free form or complexed with 3-PG. The corresponding structures were solved to high resolution (1.8 and 1.6 A, respectively) and compared. Essentially, binding 3-PG caused a 6° rotation of the N-domain in respect to the C-domain. The recombinant enzyme exhibited kinetic properties similar to those of the authentic enzyme, displaying vs 3-PG and ATP alike specific activities (about 1000 U/mg) and alike Km values (about 1mM). I47N and S320N mutant enzymes showed kcat values 3-fold lower than the wild-type enzyme. The D164V was characterized by a Km value vs 3-PG 15 times higher than that of the other enzymes studied and a catalytic efficiency 70 times lower. Finally, all mutant enzymes turned out to be highly heat unstable with respect to the wildtype enzyme, losing half of their activity after approximately 10 minutes of incubation at 37 °C. At higher temperatures, the wild-type enzyme was protected from heat inactivation by Mg-ATP or 3-PG. On the contrary, no one mutant was protect by Mg-ATP and the D164V and S320N mutants were not even protected by 3-PG. Therefore, these preliminary studies indicate that all mutations target amino acid residues located in positions primarily important for preserving the protein stability during the red cell life span.
44
Reeve, Stephanie M., Pablo Gainza, Kathleen M. Frey, Ivelin Georgiev, Bruce R. Donald, and Amy C. Anderson. "Protein design algorithms predict viable resistance to an experimental antifolate." Proceedings of the National Academy of Sciences 112, no. 3 (December 31, 2014): 749–54. http://dx.doi.org/10.1073/pnas.1411548112.
Full textAbstract:
Methods to accurately predict potential drug target mutations in response to early-stage leads could drive the design of more resilient first generation drug candidates. In this study, a structure-based protein design algorithm (K* in the OSPREY suite) was used to prospectively identify single-nucleotide polymorphisms that confer resistance to an experimental inhibitor effective against dihydrofolate reductase (DHFR) from Staphylococcus aureus. Four of the top-ranked mutations in DHFR were found to be catalytically competent and resistant to the inhibitor. Selection of resistant bacteria in vitro reveals that two of the predicted mutations arise in the background of a compensatory mutation. Using enzyme kinetics, microbiology, and crystal structures of the complexes, we determined the fitness of the mutant enzymes and strains, the structural basis of resistance, and the compensatory relationship of the mutations. To our knowledge, this work illustrates the first application of protein design algorithms to prospectively predict viable resistance mutations that arise in bacteria under antibiotic pressure.
45
Meneau, I., D. Sanglard, J. Bille, and P. M. Hauser. "Pneumocystis jiroveci Dihydropteroate Synthase Polymorphisms Confer Resistance to Sulfadoxine and Sulfanilamide in Saccharomyces cerevisiae." Antimicrobial Agents and Chemotherapy 48, no. 7 (July 2004): 2610–16. http://dx.doi.org/10.1128/aac.48.7.2610-2616.2004.
Full textAbstract:
ABSTRACT Failure of anti-Pneumocystis jiroveci prophylaxis with sulfa drugs is associated with mutations within the putative active site of the fungal dihydropteroate synthase (DHPS), an enzyme encoded by the multidomain FAS gene. This enzyme is involved in the essential biosynthesis of folic acid. The most frequent polymorphisms are two mutations leading to two amino acid changes (55Trp-Arg-57Pro to 55Ala-Arg-57Ser), observed as a single or double mutation in the same P. jiroveci isolate. In the absence of a culture method for P. jiroveci, we studied potential resistance to sulfa drugs conferred by these polymorphisms by using Saccharomyces cerevisiae as a model. Single or double mutations identical to those observed in the DHPS domain of the P. jiroveci FAS gene were introduced by in vitro site-directed mutagenesis into alleles of the S. cerevisiae FOL1 gene, which is the orthologue of the P. jiroveci FAS gene. The mutated alleles were integrated at the genomic locus in S. cerevisiae and expressed by functional complementation in a strain with a disrupted FOL1 allele. The single mutation 55Trp to 55Ala conferred resistance to sulfanilamide, whereas the single mutation 57Pro to 57Ser conferred resistance to both sulfanilamide and sulfadoxine. Both single mutations also separately conferred hypersensitivity to sulfamethoxazole and dapsone. The resistance to sulfadoxine is consistent with epidemiological data on P. jiroveci. The double mutation 55Trp-Arg-57Pro to 55Ala-Arg-57Ser conferred on S. cerevisiae a requirement for p-aminobenzoate, suggesting reduced affinity of DHPS for this substrate. This characteristic is commonly observed in mutated DHPS enzymes conferring sulfa drug resistance from other organisms. However, the double mutation conferred hypersensitivity to sulfamethoxazole, which is not in agreement with epidemiological data on P. jiroveci. Taken together, our results suggest that the DHPS polymorphisms observed in P. jiroveci confer sulfa drug resistance on this pathogen.
46
Nissley, Dwight V., Jessica Radzio, Zandrea Ambrose, Chih-Wei Sheen, Noureddine Hamamouch, Katie L. Moore, Gilda Tachedjian, and Nicolas Sluis-Cremer. "Characterization of novel non-nucleoside reverse transcriptase (RT) inhibitor resistance mutations at residues 132 and 135 in the 51 kDa subunit of HIV-1 RT." Biochemical Journal 404, no. 1 (April 26, 2007): 151–57. http://dx.doi.org/10.1042/bj20061814.
Full textAbstract:
Several rare and novel NNRTI [non-nucleoside reverse transcriptase (RT) inhibitor] resistance mutations were recently detected at codons 132 and 135 in RTs from clinical isolates using the yeast-based chimaeric TyHRT (Ty1/HIV-1 RT) phenotypic assay. Ile132 and Ile135 form part of the β7–β8 loop of HIV-1 RT (residues 132–140). To elucidate the contribution of these residues in RT structure–function and drug resistance, we constructed twelve recombinant enzymes harbouring mutations at codons 132 and 135–140. Several of the mutant enzymes exhibited reduced DNA polymerase activities. Using the yeast two-hybrid assay for HIV-1 RT dimerization we show that in some instances this decrease in enzyme activity could be attributed to the mutations, in the context of the 51 kDa subunit of HIV-1 RT, disrupting the subunit–subunit interactions of the enzyme. Drug resistance analyses using purified RT, the TyHRT assay and antiviral assays demonstrated that the I132M mutation conferred high-level resistance (>10-fold) to nevirapine and delavirdine and low-level resistance (∼2–3-fold) to efavirenz. The I135A and I135M mutations also conferred low level NNRTI resistance (∼2-fold). Subunit selective mutagenesis studies again demonstrated that resistance was conferred via the p51 subunit of HIV-1 RT. Taken together, our results highlight a specific role of residues 132 and 135 in NNRTI resistance and a general role for residues in the β7–β8 loop in the stability of HIV-1 RT.
47
Xu, Hong-Tao, Maureen Oliveira, Peter K. Quashie, Matthew McCallum, Yingshan Han, Yudong Quan, Bluma G. Brenner, and Mark A. Wainberg. "Subunit-Selective Mutational Analysis and Tissue Culture Evaluations of the Interactions of the E138K and M184I Mutations in HIV-1 Reverse Transcriptase." Journal of Virology 86, no. 16 (May 23, 2012): 8422–31. http://dx.doi.org/10.1128/jvi.00271-12.
Full textAbstract:
The emergence of HIV-1 drug resistance remains a major obstacle in antiviral therapy. M184I/V and E138K are signature mutations of clinical relevance in HIV-1 reverse transcriptase (RT) for the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and emtricitabine (FTC) and the second-generation (new) nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV), respectively, and the E138K mutation has also been shown to be selected by etravirine in cell culture. The E138K mutation was recently shown to compensate for the low enzyme processivity and viral fitness associated with the M184I/V mutations through enhanced deoxynucleoside triphosphate (dNTP) usage, while the M184I/V mutations compensated for defects in polymerization rates associated with the E138K mutations under conditions of high dNTP concentrations. The M184I mutation was also shown to enhance resistance to RPV and ETR when present together with the E138K mutation. These mutual compensatory effects might also enhance transmission rates of viruses containing these two mutations. Therefore, we performed tissue culture studies to investigate the evolutionary dynamics of these viruses. Through experiments in which E138K-containing viruses were selected with 3TC-FTC and in which M184I/V viruses were selected with ETR, we demonstrated that ETR was able to select for the E138K mutation in viruses containing the M184I/V mutations and that the M184I/V mutations consistently emerged when E138K viruses were selected with 3TC-FTC. We also performed biochemical subunit-selective mutational analyses to investigate the impact of the E138K mutation on RT function and interactions with the M184I mutation. We now show that the E138K mutation decreased rates of polymerization, impaired RNase H activity, and conferred ETR resistance through the p51 subunit of RT, while an enhancement of dNTP usage as a result of the simultaneous presence of both mutations E138K and M184I occurred via both subunits.
48
Davis, Meryl A., Marion C. Askin, and Michael J. Hynes. "Amino Acid Catabolism by an areA-Regulated Gene Encoding an l-Amino Acid Oxidase with Broad Substrate Specificity in Aspergillus nidulans." Applied and Environmental Microbiology 71, no. 7 (July 2005): 3551–55. http://dx.doi.org/10.1128/aem.71.7.3551-3555.2005.
Full textAbstract:
ABSTRACT The filamentous fungus Aspergillus nidulans can use a wide range of compounds as nitrogen sources. The synthesis of the various catabolic enzymes needed to breakdown these nitrogen sources is regulated by the areA gene, which encodes a GATA transcription factor required to activate gene expression under nitrogen-limiting conditions. The areA102 mutation results in pleiotropic effects on nitrogen source utilization, including better growth on certain amino acids as nitrogen sources. Mutations in the sarA gene were previously isolated as suppressors of the strong growth of an areA102 strain on l-histidine as a sole nitrogen source. We cloned the sarA gene by complementation of a sarA mutant and showed that it encodes an l-amino acid oxidase enzyme with broad substrate specificity. Elevated expression of this enzyme activity in an areA102 background accounts for the strong growth of these strains on amino acids that are substrates for this enzyme. Loss of function sarA mutations, which abolish the l-amino acid oxidase activity, reverse the areA102 phenotype. Growth tests with areA102 and sarA mutants show that this enzyme is the primary route of catabolism for some amino acids, while other amino acids are metabolized through alternative pathways that yield either ammonium or glutamate for growth.
49
Saumaa, Signe, Andres Tover, Mariliis Tark, Radi Tegova, and Maia Kivisaar. "Oxidative DNA Damage Defense Systems in Avoidance of Stationary-Phase Mutagenesis in Pseudomonas putida." Journal of Bacteriology 189, no. 15 (June 1, 2007): 5504–14. http://dx.doi.org/10.1128/jb.00518-07.
Full textAbstract:
ABSTRACT Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbon-starved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria.
50
Faber, Matthew S., Emily E. Wrenbeck, Laura R. Azouz, Paul J. Steiner, and Timothy A. Whitehead. "Impact of In Vivo Protein Folding Probability on Local Fitness Landscapes." Molecular Biology and Evolution 36, no. 12 (August 10, 2019): 2764–77. http://dx.doi.org/10.1093/molbev/msz184.
Full textAbstract:
Abstract It is incompletely understood how biophysical properties like protein stability impact molecular evolution and epistasis. Epistasis is defined as specific when a mutation exclusively influences the phenotypic effect of another mutation, often at physically interacting residues. In contrast, nonspecific epistasis results when a mutation is influenced by a large number of nonlocal mutations. As most mutations are pleiotropic, the in vivo folding probability—governed by basal protein stability—is thought to determine activity-enhancing mutational tolerance, implying that nonspecific epistasis is dominant. However, evidence exists for both specific and nonspecific epistasis as the prevalent factor, with limited comprehensive data sets to support either claim. Here, we use deep mutational scanning to probe how in vivo enzyme folding probability impacts local fitness landscapes. We computationally designed two different variants of the amidase AmiE with statistically indistinguishable catalytic efficiencies but lower probabilities of folding in vivo compared with wild-type. Local fitness landscapes show slight alterations among variants, with essentially the same global distribution of fitness effects. However, specific epistasis was predominant for the subset of mutations exhibiting positive sign epistasis. These mutations mapped to spatially distinct locations on AmiE near the initial mutation or proximal to the active site. Intriguingly, the majority of specific epistatic mutations were codon dependent, with different synonymous codons resulting in fitness sign reversals. Together, these results offer a nuanced view of how protein folding probability impacts local fitness landscapes and suggest that transcriptional–translational effects are as important as stability in determining evolutionary outcomes.