Journal articles: 'Structural analysis and biochemical characterization of lipases'

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Mohd Din, Mohd Hadzdee, Anusha Nair, Malihe Masomian, Mohd Shukuri Mohamad Ali, and Raja Noor Zaliha Raja Abd. Rahman. "Heterologous Expression and Characterization of Plant Lipase LIP2 from Elaeis guineensis Jacq. Oil Palm Mesocarp in Escherichia coli." Catalysts 11, no. 2 (February 12, 2021): 244. http://dx.doi.org/10.3390/catal11020244.

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In order to determine the potential of biochemical and structural features of Elaeis guineensis Jacq. oil palm mesocarp lipases, the LIP2 gene was isolated, expressed, purified and characterized through the Escherichia coli microbial recombinant system. Gene analysis of LIP2 revealed that it is composed of 1584 base pairs which are encoded in 528 amino acid residues with a molecular weight of around 57 kDa. LIP2 has distinctive lipolytic properties in terms of α/β fold and the catalytic triad for lipase. The LIP2 lipase was successfully expressed and purified from E. coli Rosetta (DE3) via affinity chromatography. The optimal temperature and pH for the lipase activity was 30 °C and a pH of 9, respectively. Stability was profoundly increased with the addition of metal ions (Ca2+, Mg2+, Mn+, and Ni+), along with organic solvents (ethanol and octanol). pNP myristate was the most suitable among all pNP esters. In biophysical characterization analysis, LIP2 has a thermal denaturing point at 66 °C, which mostly consists of random patterns (39.8%) followed by α-helix (30.3%), turns (23.8%) and β-sheet (6.2%). From the successful purification and characterization, the potential of oil palm mesocarp lipase was able to be further explored.

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Liu, Guangyuan, Xue Meng, Yujun Ren, Min Zhang, Ziqing Chen, Zhaoqi Zhang, Xuequn Pang, and Xuelian Zhang. "Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum." International Journal of Molecular Sciences 23, no. 19 (October 3, 2022): 11716. http://dx.doi.org/10.3390/ijms231911716.

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Recent studies have confirmed that chlorophyllase (CLH), a long-found chlorophyll (Chl) dephytylation enzyme for initiating Chl catabolism, has no function in leaf senescence-related Chl breakdown. Yet, CLH is considered to be involved in fruit degreening and responds to external and hormonal stimuli. The purpose of this work was to elucidate in detail the biochemical, structural properties, and gene expression of four CLHs from the Solanum lycopersicum genome so as to understand the roles of Solanum lycopersicum chlorophyllases (SlCLHs). SlCLH1/4 were the predominantly expressed CLH genes during leaf and fruit development/ripening stages, and SlCLH1 in mature green fruit was modulated by light. SlCLH1/2/3/4 contained a highly conserved GHSXG lipase motif and a Ser-Asp-His catalytic triad. We identified Ser159, Asp226, and His258 as the essential catalytic triad by site-directed mutagenesis in recombinant SlCLH1. Kinetic analysis of the recombinant enzymes revealed that SlCLH1 had high hydrolysis activities against Chl a, Chl b, and pheophytin a (Phein a), but preferred Chl a and Chl b over Phein a; SlCLH2/3 only showed very low activity to Chl a and Chl b, while SlCLH4 showed no Chl dephytylation activity. The recombinant SlCLH1/2/3 had different pH stability and temperature optimum. Removal of the predicted N-terminal processing peptide caused a partial loss of activity in recombinant SlCLH1/2 but did not compromise SlCLH3 activity. These different characteristics among SlCLHs imply that they may have different physiological functions in tomato.

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Boyko, Konstantin M., Mariya V. Kryukova, Lada E. Petrovskaya, Elena A. Kryukova, Alena Y. Nikolaeva, Dmitry A. Korzhenevsky, Galina Yu Lomakina, et al. "Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure." Biomolecules 11, no. 1 (January 5, 2021): 57. http://dx.doi.org/10.3390/biom11010057.

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The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/β hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. Km values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.

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Deng, Qiang, Jian-wei Zhai, Marie-Louise Michel, Jun Zhang, Jun Qin, Yu-ying Kong, Xin-xin Zhang, et al. "Identification and Characterization of Peptides That Interact with Hepatitis B Virus via the Putative Receptor Binding Site." Journal of Virology 81, no. 8 (December 27, 2006): 4244–54. http://dx.doi.org/10.1128/jvi.01270-06.

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ABSTRACT A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W1T2X3W4W5) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.

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Ben Hlima, Hajer, Mouna Dammak, Aida Karray, Maroua Drira, Philippe Michaud, Imen Fendri, and Slim Abdelkafi. "Molecular and Structural Characterizations of Lipases from Chlorella by Functional Genomics." Marine Drugs 19, no. 2 (January 28, 2021): 70. http://dx.doi.org/10.3390/md19020070.

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Microalgae have been poorly investigated for new-lipolytic enzymes of biotechnological interest. In silico study combining analysis of sequences homologies and bioinformatic tools allowed the identification and preliminary characterization of 14 putative lipases expressed by Chlorella vulagaris. These proteins have different molecular weights, subcellular localizations, low instability index range and at least 40% of sequence identity with other microalgal lipases. Sequence comparison indicated that the catalytic triad corresponded to residues Ser, Asp and His, with the nucleophilic residue Ser positioned within the consensus GXSXG pentapeptide. 3D models were generated using different approaches and templates and demonstrated that these putative enzymes share a similar core with common α/β hydrolases fold belonging to family 3 lipases and class GX. Six lipases were predicted to have a transmembrane domain and a lysosomal acid lipase was identified. A similar mammalian enzyme plays an important role in breaking down cholesteryl esters and triglycerides and its deficiency causes serious digestive problems in human. More structural insight would provide important information on the enzyme characteristics.

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Soloski, M. J., A. Lattimore, D. Hereld, J. L. Krakow, M. G. Low, and G. Einhorn. "Further characterization of the membrane anchor found on the tissue-specific class I molecule Qa2." Journal of Immunology 140, no. 11 (June 1, 1988): 3858–66. http://dx.doi.org/10.4049/jimmunol.140.11.3858.

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Abstract Previous studies have determined that various Qa2 serologic determinants can be removed from the surface of spleen cells by treatment with a phospholipase C. Our studies have determined that the class I molecule Qa2, expressed on the surface of spleen cells and activated T cells, behaves as an integral membrane protein based on its ability to associate with detergent micelles. Studies utilizing two purified phospholipase C have revealed that although most (90 to 95%) of the Qa2 molecules expressed on the surface of resting spleen cells are released as intact 40-kDa polypeptides associated with beta 2-microglobulin, activated T cells contain a major cell subpopulation expressing lipase-resistant Qa2 molecules. Flow cytometric analysis revealed that L3T4+-activated T cells expressed lipase-sensitive Qa2 molecules, whereas Lyt-2+ cells express lipase-resistant forms of the Qa2 molecule. The relationship between the secreted form of the Qa2 molecule and the lipase-generated soluble Qa2 molecule was investigated. Based on SDS-PAGE analysis, the secreted Qa2 molecules has a Mr of 39 kDa whereas the cell surface form released from either resting spleen or activated T cells by phosphatidylinositol-specific phospholipase C has a Mr of approximately equal to 40 kDa. Furthermore, the secreted Qa2 molecule lacks an epitope, cross-reacting determinant, often present on lipase-solubilized cell surface molecules. Thus, based on serologic and biochemical criteria, the soluble Qa2 molecules generated by an exogenous phospholipase C and the secreted Qa2 molecule are structurally distinct.

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Guo, Chenchen, Rikuan Zheng, Ruining Cai, Chaomin Sun, and Shimei Wu. "Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium." Microorganisms 9, no. 4 (April 10, 2021): 802. http://dx.doi.org/10.3390/microorganisms9040802.

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The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes.

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Kumari, Arti, Ved Vrat Verma, and Rani Gupta. "Comparative biochemical characterization and in silico analysis of novel lipases Lip11 and Lip12 with Lip2 from Yarrowia lipolytica." World Journal of Microbiology and Biotechnology 28, no. 11 (August 31, 2012): 3103–11. http://dx.doi.org/10.1007/s11274-012-1120-4.

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Turner, James M., Nicholas A. Larsen, Amrik Basran, Carlos F. Barbas, Neil C. Bruce, Ian A. Wilson, and Richard A. Lerner. "Biochemical Characterization and Structural Analysis of a Highly Proficient Cocaine Esterase†,‡." Biochemistry 41, no. 41 (October 2002): 12297–307. http://dx.doi.org/10.1021/bi026131p.

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Malavaki, Christina J., Achilleas D. Theocharis, Fotini N. Lamari, Ioannis Kanakis, Theodore Tsegenidis, George N. Tzanakakis, and Nikos K. Karamanos. "Heparan sulfate: biological significance, tools for biochemical analysis and structural characterization." Biomedical Chromatography 25, no. 1-2 (December 28, 2010): 11–20. http://dx.doi.org/10.1002/bmc.1536.

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Alabdalall, Amira Hassan, Norah Ayad ALanazi, Sumayh A. Aldakeel, Sayed AbdulAzeez, and J. Francis Borgio. "Molecular, physiological, and biochemical characterization of extracellular lipase production by Aspergillus niger using submerged fermentation." PeerJ 8 (July 7, 2020): e9425. http://dx.doi.org/10.7717/peerj.9425.

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Background Extracellular production of fungal lipases especially the lipases obtained from the Aspergilli has gained immense interest in recent years due to its diverse biotechnological applications. In this study, we focused on determining the fermentation parameters required for the optimal lipase production. Methods A total of 256 fungal isolates were obtained from oil seeds. From each genus, one isolate was selected to evaluate lipase production using phenol red and tributyrin plate assays. Lipase activity was estimated using the spectrophotometric pNPP hydrolysis assay. The highest lipase producer isolates were identified using 18S ribosomal RNA gene sequencing. The genetic variability was determined by random amplified polymorphic DNA (RAPD) analysis and the dendrogram was constructed using the unweighted pair group method with arithmetic averages method. The isolates were examined in a submerged fermentation culture (Smf) to measure the effect of temperature, pH, incubation time, carbon source, nitrogen source, inoculum volume, and lipid source on lipase production. Results Eleven isolates belonging to the genus Aspergillus were analyzed for lipase production where they were found to be the highest lipase producers among various fungal genera. All the tested isolates were identified as A. niger using 18s rRNA sequencing. Genetic diversity was evaluated among all of the studied A. niger isolates using RAPD primers. The RAPD primers were used to amplify 285 loci, of which five were polymorphic (1.75%) and seven were monomorphic (2.45%). Thus, a high level of genetic diversity was observed among all isolates. The tributyrin test and the lipase activity assay identified five strains of A. niger as high lipase producers, and their optimal enzyme activities were 709.74, 532.54, 735.64, 794.62, and 787.69 U/ml. The optimal conditions for lipase production were as follows: 40 °C, pH 7.5, 1% fructose as the carbon source, 1% yeast extract as the nitrogen source, 2% palm oil, 2.5 × 107 spores/ml suspension, and 3 days of incubation. Conclusions The current study provides a comprehensive characterization of the optimal conditions, which are essential to enhance lipase production in five A. niger isolates.

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Nezammahalleh, Hassan, Faezeh Ghanati, Shima Rezaei, Mohsin Ali Badshah, Joobee Park, Naseem Abbas, and Ahsan Ali. "Biochemical Interactions through Microscopic Techniques: Structural and Molecular Characterization." Polymers 14, no. 14 (July 13, 2022): 2853. http://dx.doi.org/10.3390/polym14142853.

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Many researchers and scientists have contributed significantly to provide structural and molecular characterizations of biochemical interactions using microscopic techniques in the recent decade, as these biochemical interactions play a crucial role in the production of diverse biomaterials and the organization of biological systems. The properties, activities, and functionalities of the biomaterials and biological systems need to be identified and modified for different purposes in both the material and life sciences. The present study aimed to review the advantages and disadvantages of three main branches of microscopy techniques (optical microscopy, electron microscopy, and scanning probe microscopy) developed for the characterization of these interactions. First, we explain the basic concepts of microscopy and then the breadth of their applicability to different fields of research. This work could be useful for future research works on biochemical self-assembly, biochemical aggregation and localization, biological functionalities, cell viability, live-cell imaging, material stability, and membrane permeability, among others. This understanding is of high importance in rapid, inexpensive, and accurate analysis of biochemical interactions.

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Yuan, Shuo-Fu, Tzu-Hui Wu, Hsiao-Lin Lee, Han-Yu Hsieh, Wen-Ling Lin, Barbara Yang, Chih-Kang Chang, et al. "Biochemical Characterization and Structural Analysis of a Bifunctional Cellulase/Xylanase fromClostridium thermocellum." Journal of Biological Chemistry 290, no. 9 (January 9, 2015): 5739–48. http://dx.doi.org/10.1074/jbc.m114.604454.

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Tang, Wei, Dongming Lan, Zexin Zhao, Shuang Li, Xiuting Li, and Yonghua Wang. "A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study." International Journal of Molecular Sciences 20, no. 3 (February 12, 2019): 780. http://dx.doi.org/10.3390/ijms20030780.

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Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.

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BERGERON, Alain, Hélène LaRUE, and Yves FRADET. "Biochemical analysis of a bladder-cancer-associated mucin: structural features and epitope characterization." Biochemical Journal 321, no. 3 (February 1, 1997): 889–96. http://dx.doi.org/10.1042/bj3210889.

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Three monoclonal antibodies (mAbs), M344, M300 and M75, were shown to define a unique tumour-associated antigen (TAA) of superficial bladder tumours. The antigenic determinants are expressed on a very-high-molecular-mass component and, in about 50% of the positive samples, one determinant is also detected on a 62 kDa molecular species, observed only under reducing conditions. The objectives of the present study were to characterize further this TAA by analysing (1) the biochemical nature of the epitopes recognized by the three mAbs, and (2) the biochemical and structural features of the molecule bearing them. The antigenicity was resistant to heat denaturation, trypsin and α-chymotrypsin treatments but highly sensitive to papain and Pronase digestion. NaIO4 oxidation decreased reactivity to mAbs M344 and M300 but enhanced reactivity to mAb M75. The three determinants were insensitive to β-galactosidase and α-l-fucosidase but were sensitive to Vibrio choleraeneuraminidase. None of the three mAbs reacted with ovine, bovine or porcine submaxillary mucins. Deglycosylation with O-glycosidase or trifluoromethanesulphonic acid completely abolished the reactivity of the mAbs whereas N-glycosidase F deglycosylation had no appreciable effect. The presence on the molecule of cryptic Galβ(1→3)GalNAc as a major core disaccharide was demonstrated by a heterologous sandwich assay using mAb M75 and peanut agglutinin. Thiol reduction using β-mercaptoethanol increased mobility of the high-molecular-mass component in polyacrylamide gels. We thus conclude that mAbs M344 and M300 react with sialylated carbohydrate epitopes, and mAb M75 reacts with a partially cryptic and periodate-resistant sialylated epitope expressed on a typical secreted high-molecular-mass oligomeric mucin which we named MAUB for mucin antigen of the urinary bladder.

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Parwani, Anil V., Linda J. Saif, and Shien-Young Kang. "Biochemical characterization of porcine enteric calicivirus: analysis of structural and nonstructural viral proteins." Archives of Virology 112, no. 1-2 (March 1990): 41–53. http://dx.doi.org/10.1007/bf01348984.

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Williams, Robert H., Mashouf Shaykh, Sarosh Ahmed, Theodore Musiala, Nadine Bazilinski, George Dunea, and Alvin Dubin. "Purification and biochemical characterization of xanthopterin from patients with chronic renal failure. II. Biochemical elucidation and structural analysis." Clinical Biochemistry 24, no. 5 (October 1991): 407–15. http://dx.doi.org/10.1016/s0009-9120(05)80016-5.

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Usachev, Konstantin S., Bulat F. Fatkhullin, Evelina A. Klochkova, Aynur K. Miftakhov, Alexander A. Golubev, Aidar G. Bikmullin, Liliya I. Nurullina, et al. "Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization." Journal of Structural Biology 209, no. 1 (January 2020): 107408. http://dx.doi.org/10.1016/j.jsb.2019.107408.

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Williams, M. G., J. Wilsher, P. Nugent, A. Mills, V. Dhanaraj, M. Fabry, J. Sedlacek, et al. "Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin." Protein Engineering Design and Selection 10, no. 9 (September 1, 1997): 991–97. http://dx.doi.org/10.1093/protein/10.9.991.

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Scaglione, Jamie B, David L Akey, Rachel Sullivan, Jeffrey D Kittendorf, Christopher M Rath, Eung-Soo Kim, Janet L Smith, and David H Sherman. "Biochemical and Structural Characterization of the Tautomycetin Thioesterase: Analysis of a Stereoselective Polyketide Hydrolase." Angewandte Chemie International Edition 49, no. 33 (July 9, 2010): 5726–30. http://dx.doi.org/10.1002/anie.201000032.

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Scaglione, Jamie B, David L Akey, Rachel Sullivan, Jeffrey D Kittendorf, Christopher M Rath, Eung-Soo Kim, Janet L Smith, and David H Sherman. "Biochemical and Structural Characterization of the Tautomycetin Thioesterase: Analysis of a Stereoselective Polyketide Hydrolase." Angewandte Chemie 122, no. 33 (July 9, 2010): 5862–66. http://dx.doi.org/10.1002/ange.201000032.

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Le, Ly Thi Huong Luu, Wanki Yoo, Changwoo Lee, Ying Wang, Sangeun Jeon, Kyeong Kyu Kim, Jun Hyuck Lee, and T. Doohun Kim. "Molecular Characterization of a Novel Cold-Active Hormone-Sensitive Lipase (HaHSL) from Halocynthiibacter Arcticus." Biomolecules 9, no. 11 (November 5, 2019): 704. http://dx.doi.org/10.3390/biom9110704.

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Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the catalytic domains of human HSLs, have received great interest due to their uses in the preparation of highly valuable biochemicals, such as drug intermediates or chiral building blocks. Here, a novel cold-active HSL from Halocynthiibacter arcticus (HaHSL) was examined and its enzymatic properties were investigated using several biochemical and biophysical methods. Interestingly, HaHSL acted on a large variety of substrates including tertiary alcohol esters and fish oils. Additionally, this enzyme was highly tolerant to high concentrations of salt, detergents, and glycerol. Furthermore, immobilized HaHSL retained its activity for up to six cycles of use. Homology modeling suggested that aromatic amino acids (Trp23, Tyr74, Phe78, Trp83, and Phe245) in close proximity to the substrate-binding pocket were important for enzyme activity. Mutational analysis revealed that Tyr74 played an important role in substrate specificity, thermostability, and enantioselectivity. In summary, the current study provides an invaluable insight into the novel cold-active HaHSL from H. arcticus, which can be efficiently and sustainably used in a wide range of biotechnological applications.

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Erlandson, M. A., and D. A. Streett. "ENTOMOPOXVIRUSES ASSOCIATED WITH GRASSHOPPERS AND LOCUSTS: BIOCHEMICAL CHARACTERIZATION." Memoirs of the Entomological Society of Canada 129, S171 (1997): 131–46. http://dx.doi.org/10.4039/entm129171131-1.

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AbstractEntomopoxviruses (EPVs) are large DNA viruses with structural similarities to vertebrate poxviruses. EPV virions are occluded in large (3–15 μm in diameter) proteinaceous occlusion bodies (OBs). To date, EPVs are reported from 15 species of grasshoppers and locusts. The current information on the biochemical characterization of these EPVs is summarized in our review. The DNA genomes of grasshopper and locust EPVs analysed to date have a G+C ratio of approximately 18.5% and genome size estimates generated by various methods range from 180 to 194 kilobase pairs (kbp). Restriction endonuclease enzyme analysis of a number of grasshopper and locust EPV DNAs shows the virus isolates to be distinct and the technique will be useful in identifying virus isolates. The structural proteins of certain grasshopper EPVs have also been analysed. Forty to 50 polypeptides ranging in molecular weight from 12 to 200 kilodaltons (kDa) have been detected by SDS-PAGE analysis of virions released from OBs and the polypeptide profiles are distinct for many of the virus isolates. The proteinaceous matrix of the OB of EPVs contains one predominant protein referred to as spheroidin. The spheroidin protein from most grasshopper EPVs is approximately the same molecular weight, 107 kDa, when analysed by SDS-PAGE. As with other groups of occluded insect viruses, grasshopper EPVs have a protease activity associated with OBs derived from infected insects. The possible role of this protease activity in the infection cycle is discussed. Finally, the role of various molecular techniques for the detection and identification of EPV infections in laboratory and field populations of grasshoppers and locusts is discussed. The development of EPV-specific monoclonal antibodies and DNA hybridization probes for the detection of virus infections is reviewed. As well, the possible use of polymerase chain reaction and randomly amplified polymorphic DNA fingerprinting techniques for the detection and identification of EPV infections is discussed.

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Guo, Peng-Chao, Qian Wang, Zhan Wang, Zhaoming Dong, Huawei He, and Ping Zhao. "Biochemical characterization and functional analysis of invertase Bmsuc1 from silkworm, Bombyx mori." International Journal of Biological Macromolecules 107 (February 2018): 2334–41. http://dx.doi.org/10.1016/j.ijbiomac.2017.10.118.

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Kascakova, Barbora, Jan Kotal, Larissa Almeida Martins, Zuzana Berankova, Helena Langhansova, Eric Calvo, Joel A. Crossley, et al. "Structural and biochemical characterization of the novel serpin Iripin-5 from Ixodes ricinus." Acta Crystallographica Section D Structural Biology 77, no. 9 (August 23, 2021): 1183–96. http://dx.doi.org/10.1107/s2059798321007920.

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Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5–protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

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Chang, Changsoo, Hyun Kyu Song, Byung Chul Park, Dae-Sil Lee, and Se Won Suh. "A thermostable xylose isomerase fromThermus caldophilus: biochemical characterization, crystallization and preliminary X-ray analysis." Acta Crystallographica Section D Biological Crystallography 55, no. 1 (January 1, 1999): 294–96. http://dx.doi.org/10.1107/s0907444998009019.

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Cieślak, Jolanta, Akimasa Miyanaga, Makoto Takaishi, Fumitaka Kudo, and Tadashi Eguchi. "Functional and structural characterization of IdnL7, an adenylation enzyme involved in incednine biosynthesis." Acta Crystallographica Section F Structural Biology Communications 75, no. 4 (April 1, 2019): 299–306. http://dx.doi.org/10.1107/s2053230x19002863.

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Adenylation enzymes play an important role in the selective incorporation of the cognate carboxylate substrates in natural product biosynthesis. Here, the biochemical and structural characterization of the adenylation enzyme IdnL7, which is involved in the biosynthesis of the macrolactam polyketide antibiotic incednine, is reported. Biochemical analysis showed that IdnL7 selects and activates several small amino acids. The structure of IdnL7 in complex with an L-alanyl-adenylate intermediate mimic, 5′-O-[N-(L-alanyl)sulfamoyl]adenosine, was determined at 2.1 Å resolution. The structure of IdnL7 explains the broad substrate specificity of IdnL7 towards small L-amino acids.

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Nishio, Kazuya, and Tsunehiro Mizushima. "Structural and biochemical characterization of mitochondrial citrate synthase 4 from Arabidopsis thaliana." Acta Crystallographica Section F Structural Biology Communications 76, no. 3 (March 1, 2020): 109–15. http://dx.doi.org/10.1107/s2053230x20001521.

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Citrate synthase (CS) catalyzes the conversion of oxaloacetate and acetyl coenzyme A into citrate and coenzyme A in the mitochondrial tricarboxylic acid (TCA) cycle. In plants, mitochondrial metabolism, including the TCA cycle, occurs in interaction with photosynthetic metabolism. The controlled regulation of several enzymes in the TCA cycle, such as CS, is important in plants. Here, the first crystal structure of a plant mitochondrial CS, CSY4 from Arabidopsis thaliana (AtCSY4), has been determined. Structural comparison of AtCSY4 with mitochondrial CSs revealed a high level of similarity. Inhibition analysis showed a similar manner of inhibition as in mitochondrial CSs. The effect of oxidation on one of a pair of cysteine residues in AtCSY4 was speculated upon based on the folded structure.

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Sisu, Ioana, Valentina Udrescu, Corina Flangea, Sorin Tudor, Nicolae Dinca, Lucian Rusnac, Alina Zamfir, and Eugen Sisu. "Synthesis and structural characterization of amino-functionalized polysaccharides." Open Chemistry 7, no. 1 (March 1, 2009): 66–73. http://dx.doi.org/10.2478/s11532-008-0090-8.

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AbstractA variety of carbohydrates, in particular polysaccharides can be subjected to chemical modification to obtain derivatives with amphiphilic properties, which enable biochemical or biological reactions at the polymer surface. In the present work, a polydisperse maltodextrin mixture of average molecular weight 3000 was coupled with 1,6-hexamethylenediamine (HMD) via reductive amination reaction. Resulting products were characterized by thermal analysis and positive nanoelectrospray quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Both thermal analysis and MS screening confirmed the formation of the HMD-polysaccharide coupling products. Moreover, HMD-linked polysaccharide chains containing 2 to 26 glucose building blocks were identified by nanoESI Q-TOF MS. MS/MS fragmentation using collision-induced dissociation (CID) at low ion acceleration energies provided strong evidence for HMD-maltodextrin linkage formation and the set of sequence ions diagnostic for the composition and structure of a HMD-linked chain containing 18 glucose residues.

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Chiou, Shyh-Horng, Chin-Chun Hung, and Chou-Wen Lin. "Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon δ-crystallin." Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1160, no. 3 (December 1992): 317–24. http://dx.doi.org/10.1016/0167-4838(92)90094-t.

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Golda, Mária, János András Mótyán, Katalin Nagy, Krisztina Matúz, Tibor Nagy, and József Tőzsér. "Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)." Biomolecules 10, no. 7 (July 6, 2020): 1004. http://dx.doi.org/10.3390/biom10071004.

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The human retroviral-like aspartic protease 1 (ASPRV1) is a mammalian retroviral-like enzyme that catalyzes a critical proteolytic step during epidermal differentiation; therefore, it is also referred to as skin-specific aspartic protease (SASPase). Neutrophil granulocytes were also found recently to express ASPRV1 that is involved in the progression of acute chronic inflammation of the central nervous system, especially in autoimmune encephalomyelitis. Thus, investigation of ASPRV1 is important due to its therapeutic or diagnostic potential. We investigated the structural characteristics of ASPRV1 by homology modeling; analysis of the proposed structure was used for interpretation of in vitro specificity studies. For in-vitro characterization, activities of SASP28 and SASP14 enzyme forms were measured using synthetic oligopeptide substrates. We demonstrated that self-processing of SASP28 precursor causes autoactivation of the protease. The highest activity was measured for GST-SASP14 at neutral pH and at high ionic strength, and we proved that pepstatin A and acetyl-pepstatin can also inhibit the protease. In agreement with the structural characteristics, the relatively lower urea dissociation constant implied lower dimer stability of SASP14 compared to that of HIV-1 protease. The obtained structural and biochemical characteristics support better understanding of ASPRV1 function in the skin and central nervous system.

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Liu, Dandan, Gabe Small, Judd Hultquist, Nevan Krogan, Joyti Batra, Christopher Basler, Daisy Leung, and Gaya Amarasinghe. "Biochemical and structural characterization of a host protein that binds Ebola VP30." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 182.32. http://dx.doi.org/10.4049/jimmunol.200.supp.182.32.

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Abstract Ebola and Marburg viruses are negative sense RNA viruses that can cause high case fatality rates during outbreaks. Approximately 19 kb genome encodes for 7 open reading frames. During virus replication, 7–10 multifunctional proteins are expressed, but viral replication and pathogenesis also require numerous cellular proteins. While the need for host factors in virus replication and pathogenesis has been long appreciated, our understanding of key host-virus interactions during filoviral infection and replication remain incomplete. In order to address this limitation, our collaborators recently performed a proteomic analysis, which identified RBBP6, an E3 ubiquitin ligase, as a cellular interactor of Ebola VP30 (eVP30). eVP30 is a viral protein critical for transcription initiation. In this study, we generated a series of recombinant RBBP6 and eVP30 truncation constructs. We performed in vitro pull-down assays to validate the initial proteomic identification of the eVP30/RBBP6 protein-protein interaction and defined key regions within RBBP6 that are critical for eVP30 binding. Furthermore, using purified eVP30 protein, we generated crystals with a RBBP6 binding peptide and refined X-ray diffraction data to define a series of key residues on the interface of eVP30/RBBP6. Hence, this study provides insights on host-viral interface that modulates viral pathogenesis and defines a novel target for potential development of antiviral therapeutics that target filoviruses.

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Yang, Guojun, Wei Chen, Haidong Tan, Kuikui Li, Junyan Li, and Heng Yin. "Biochemical characterization and evolutionary analysis of a novel pectate lyase from Aspergillus parasiticus." International Journal of Biological Macromolecules 152 (June 2020): 180–88. http://dx.doi.org/10.1016/j.ijbiomac.2020.02.279.

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Shin, Woo-Ri, Hyun-Ju Um, Young-Chang Kim, Sun Chang Kim, Byung-Kwan Cho, Ji-Young Ahn, Jiho Min, and Yang-Hoon Kim. "Biochemical characterization and molecular docking analysis of novel esterases from Sphingobium chungbukense DJ77." International Journal of Biological Macromolecules 168 (January 2021): 403–11. http://dx.doi.org/10.1016/j.ijbiomac.2020.12.077.

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Aparaschivei, Diana, Anamaria Todea, August E. Frissen, Valentin Badea, Gerlinde Rusu, Eugen Sisu, Maria Puiu, Carmen G. Boeriu, and Francisc Peter. "Enzymatic synthesis and characterization of novel terpolymers from renewable sources." Pure and Applied Chemistry 91, no. 3 (March 26, 2019): 397–408. http://dx.doi.org/10.1515/pac-2018-1015.

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Abstract 2,5-Furandicarboxylic acid and itaconic acid are both important biobased platform chemicals and their terpolymer with 1,6-hexanediol (HDO) can be the starting point for a new class of reactive polyesters, with important applications. The green synthetic route developed in this study involves a biocatalytic condensation polymerization reaction of dimethyl furan-2,5-dicarboxylate (DMFDC) and dimethyl itaconate (DMI) with HDO in toluene at 80°C, using commercial immobilized lipases from Candida antarctica B. In the best conditions, the formed polymer product was isolated with more than 80% yield, containing about 85% terpolymer with average molecular mass of about 1200 (Mn, calculated from MALDI-TOF MS data) and 15% DMFDC_HDO copolymer. Considering the higher reactivity of DMFDC, the composition of the synthesized polymer can be directed by adjusting the molar ratio of DMFDC and DMI, as well as by extending the reaction time. Structural analysis by NMR demonstrated the regioselective preference for the carbonyl group from DMI adjacent to the methylene group. The biocatalyst was successfully reused in multiple reaction cycles.

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Bhatt, Kandarp, Sangeeta Lal, R. Srinivasan, and Bhumika Joshi. "Molecular analysis of Bacillus velezensis KB 2216, purification and biochemical characterization of alpha-amylase." International Journal of Biological Macromolecules 164 (December 2020): 3332–39. http://dx.doi.org/10.1016/j.ijbiomac.2020.08.205.

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Laskar, Rafiul Amin, Amaj Ahmed Laskar, Aamir Raina, Samiullah Khan, and Hina Younus. "Induced mutation analysis with biochemical and molecular characterization of high yielding lentil mutant lines." International Journal of Biological Macromolecules 109 (April 2018): 167–79. http://dx.doi.org/10.1016/j.ijbiomac.2017.12.067.

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Borgianni, Luisa, Filomena De Luca, Maria Cristina Thaller, Yunsop Chong, Gian Maria Rossolini, and Jean-Denis Docquier. "Biochemical Characterization of the POM-1 Metallo-β-Lactamase from Pseudomonas otitidis." Antimicrobial Agents and Chemotherapy 59, no. 3 (December 15, 2014): 1755–58. http://dx.doi.org/10.1128/aac.03843-14.

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ABSTRACTThe POM-1 metallo-β-lactamase is a subclass B3 resident enzyme produced byPseudomonas otitidis, a pathogen causing otic infections. The enzyme was overproduced inEscherichia coliBL21(DE3), purified by chromatography, and subjected to structural and functional analysis. The purified POM-1 is a tetrameric enzyme of broad substrate specificity with higher catalytic activities with penicillins and carbapenems than with cephalosporins.

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Shcherbakova, Tatiana A., Semen M. Baldin, Mikhail S. Shumkov, Irina V. Gushchina, Dmitry K. Nilov, and Vytas K. Švedas. "Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis." Acta Naturae 14, no. 2 (July 21, 2022): 93–97. http://dx.doi.org/10.32607/actanaturae.11713.

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Transketolase, an enzyme of the pentose phosphate pathway, plays an important role in the functioning of mycobacteria. Using plasmid pET-19b carrying the Rv1449c gene of transketolase from Mycobacterium tuberculosis and an additional histidine tag, we isolated and purified recombinant transketolase and determined the conditions for obtaining the apoform of the protein. The Michaelis constants were evaluated for the thiamine diphosphate cofactor in the presence of magnesium and calcium ions. We found that the affinity of mycobacterial transketolase for thiamine diphosphate is by three orders of magnitude lower than that of the human enzyme. Analysis of the structural organization of the active centers of homologous enzymes showed that this difference is due to a replacement of lysine residues by less polar amino acid residues.

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Davies, Jonathan R., Geoffrey Masuyer, and Pål Stenmark. "Structural and Biochemical Characterization of Botulinum Neurotoxin Subtype B2 Binding to Its Receptors." Toxins 12, no. 9 (September 17, 2020): 603. http://dx.doi.org/10.3390/toxins12090603.

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Botulinum neurotoxins (BoNTs) can be used therapeutically to treat a wide range of neuromuscular and neurological conditions. A collection of natural BoNT variants exists which can be classified into serologically distinct serotypes (BoNT/B), and further divided into subtypes (BoNT/B1, B2, …). BoNT subtypes share a high degree of sequence identity within the same serotype yet can display large variation in toxicity. One such example is BoNT/B2, which was isolated from Clostridium botulinum strain 111 in a clinical case of botulism, and presents a 10-fold lower toxicity than BoNT/B1. In an effort to understand the molecular mechanisms behind this difference in potency, we here present the crystal structures of BoNT/B2 in complex with the ganglioside receptor GD1a, and with the human synaptotagmin I protein receptor. We show, using receptor-binding assays, that BoNT/B2 has a slightly higher affinity for GD1a than BoNT/B1, and confirm its considerably weaker affinity for its protein receptors. Although the overall receptor-binding mechanism is conserved for both receptors, structural analysis suggests the lower affinity of BoNT/B2 is the result of key substitutions, where hydrophobic interactions important for synaptotagmin-binding are replaced by polar residues. This study provides a template to drive the development of future BoNT therapeutic molecules centered on assessing the natural subtype variations in receptor-binding that appears to be one of the principal stages driving toxicity.

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Cho, Hye Yeon, Mi Sun Nam, Ho Jeong Hong, Wan Seok Song, and Sung-il Yoon. "Structural and Biochemical Analysis of the Furan Aldehyde Reductase YugJ from Bacillus subtilis." International Journal of Molecular Sciences 23, no. 3 (February 8, 2022): 1882. http://dx.doi.org/10.3390/ijms23031882.

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NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) participates in a wide range of physiologically important cellular processes by reducing aldehydes or oxidizing alcohols. Among AAOR substrates, furan aldehyde is highly toxic to microorganisms. To counteract the toxic effect of furan aldehyde, some bacteria have evolved AAOR that converts furan aldehyde into a less toxic alcohol. Based on biochemical and structural analyses, we identified Bacillus subtilis YugJ as an atypical AAOR that reduces furan aldehyde. YugJ displayed high substrate specificity toward 5-hydroxymethylfurfural (HMF), a furan aldehyde, in an NADPH- and Ni2+-dependent manner. YugJ folds into a two-domain structure consisting of a Rossmann-like domain and an α-helical domain. YugJ interacts with NADP and Ni2+ using the interdomain cleft of YugJ. A comparative analysis of three YugJ structures indicated that NADP(H) binding plays a key role in modulating the interdomain dynamics of YugJ. Noticeably, a nitrate ion was found in proximity to the nicotinamide ring of NADP in the YugJ structure, and the HMF-reducing activity of YugJ was inhibited by nitrate, providing insights into the substrate-binding mode of YugJ. These findings contribute to the characterization of the YugJ-mediated furan aldehyde reduction mechanism and to the rational design of improved furan aldehyde reductases for the biofuel industry.

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BLACKBURN, Paul E., Clare V. SIMPSON, Robert J. B. NIBBS, Maureen O'HARA, Rhona BOOTH, Jemma POULOS, Neil W. ISAACS, and Gerard J. GRAHAM. "Purification and biochemical characterization of the D6 chemokine receptor." Biochemical Journal 379, no. 2 (April 15, 2004): 263–72. http://dx.doi.org/10.1042/bj20031266.

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There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, autoimmune and inflammatory disorders, and cancer. Hampering such studies is the lack of accurate three-dimensional structural models of these molecules. The CC-chemokine receptor D6 is expressed at exceptionally high levels in heterologous transfectants. Here we report the purification and biochemical characterization of milligram quantities of D6 protein from relatively small cultures of transfected mammalian cells. Importantly, purified D6 retains full functional activity, shown by displaceable binding of 125I-labelled MIP-1β (macrophage inflammatory protein-1β) and by complete binding of the receptor to a MIP-1α affinity column. In addition, we show that D6 is decorated on the N-terminus by N-linked glycosylation. Mutational analysis reveals that this glycosylation is dispensable for ligand binding and high expression in transfected cells. Metabolic labelling has revealed the receptor to also be sulphated and phosphorylated. Phosphorylation is ligand independent and is not enhanced by ligand binding and internalization, suggesting similarities with the viral chemokine receptor homologue US28. Like US28, an analysis of the full cellular complement of D6 in transfected cells indicates that >80% is found associated with intracellular vesicular structures. This may account for the high quantities of D6 that can be synthesized in these cells. These unusual properties of D6, and the biochemical characterization described here, leads the way towards work aimed at generating the three-dimensional structure of this seven-transmembrane-spanning receptor.

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Ker, De-Sheng, Sze Lei Pang, Noor Farhan Othman, Sekar Kumaran, Ee Fun Tan, Thiba Krishnan, Kok Gan Chan, Roohaida Othman, Maizom Hassan, and Chyan Leong Ng. "Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase." PeerJ 5 (February 28, 2017): e2961. http://dx.doi.org/10.7717/peerj.2961.

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Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, addition of 15% (v/v) glycerol to the protein purification buffer and removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.

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Urbański, Linda J., Andrea Angeli, Vasyl V. Mykuliak, Latifeh Azizi, Marianne Kuuslahti, Vesa P. Hytönen, Claudiu T. Supuran, and Seppo Parkkila. "Biochemical and structural characterization of beta-carbonic anhydrase from the parasite Trichomonas vaginalis." Journal of Molecular Medicine 100, no. 1 (October 15, 2021): 115–24. http://dx.doi.org/10.1007/s00109-021-02148-1.

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Abstract Trichomonas vaginalis is a unicellular parasite and responsible for one of the most common sexually transmittable infections worldwide, trichomoniasis. Carbonic anhydrases (CAs) are enzymes found in all lifeforms and are known to play a vital role in many biochemical processes in organisms including the maintenance of acid–base homeostasis. To date, eight evolutionarily divergent but functionally convergent forms of CAs (α, β, γ, δ, ζ, η, θ, and ι) have been discovered. The human genome contains only α-CAs, whereas many clinically significant pathogens express only β-CAs and/or γ-CAs. The characterization of pathogenic β- and γ-CAs provides important knowledge for targeting these biomolecules to develop novel anti-invectives against trichomoniasis. Here, we report the recombinant production and characterization of the second β-CA of T. vaginalis (TvaCA2). Light scattering analysis revealed that TvaCA2 is a dimeric protein, which was further supported with in silico modeling, suggesting similar structures between TvaCA2 and the first β-CA of T. vaginalis (TvaCA1). TvaCA2 exhibited moderate catalytic activity with the following kinetic parameters: kcat of 3.8 × 105 s−1 and kcat/KM of 4.4 × 107 M−1 s−1. Enzyme activity inhibition was studied with a set of clinically used sulfonamides and sulfonamide derivates. Twenty-seven out of the 39 compounds resulted in inhibition with a nanomolar range. These initial results encourage for future work entailing the design of more potent inhibitors against TvaCA2, which may provide new assets to fight trichomoniasis. Key messages • Protozoan parasite Trichomonas vaginalis has two β-carbonic anhydrases (TvaCA1/2). • TvaCA1/TvaCA2 represents promising targets for antitrichomonal drug development. • TvaCA2 is a dimer of 20.3 kDa and possesses moderate catalytic activity. • The most efficient inhibitor was clinical drug acetazolamide with KI of 222.9 nM. • The 39 tested sulfonamides form the basis for the design of more potent inhibitors.

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Richmond, Gregory S., and Terry K. Smith. "The role and characterization of phospholipase A1 in mediating lysophosphatidylcholine synthesis in Trypanosoma brucei." Biochemical Journal 405, no. 2 (June 27, 2007): 319–29. http://dx.doi.org/10.1042/bj20070193.

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Lysophospholipids are ubiquitous intermediates in a variety of metabolic and signalling pathways in eukaryotic cells. We have reported recently that lysoglycerophosphatidylcholine (lyso-GPCho) synthesis in the insect form of the ancient eukaryote Trypanosoma brucei is mediated by a novel phospholipase A1 (TbPLA1). In the present study, we show that despite equal levels of TbPLA1 gene expression in wild-type insect and bloodstream trypomastigotes, both TbPLA1 enzyme levels and lysoGPCho metabolites are approx. 3-fold higher in the bloodstream form. Both of these parasite stages synthesize identical molecular species of lysoGPCho. TbPLA1 null mutants in the bloodstream form of the parasite are viable, but are deficient in lysoGPCho synthesis, a defect that can be overcome by the expression of an ectopic copy of TbPLA1. The biochemical attributes of TbPLA1-mediated lysoGPCho synthesis were examined in vitro using recombinant TbPLA1. Although TbPLA1 possesses an active-site serine residue, it is insensitive to serine-modifying reagents, such as di-isopropyl fluorophosphate and PMSF, a characteristic shared by lipases that possess lid-sheltered catalytic triads. TbPLA1 does not require metal co-factors for activity, but it does require interfacial activation prior to catalysis. Results from size-exclusion chromatography and binding kinetics analysis revealed that TbPLA1 activation by Triton X-100/GPCho mixed micelle surfaces was not specific and did not require the pre-formation of a specific enzyme–substrate complex to achieve surface binding.

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Hesseler, Martin, Xenia Bogdanović, Aurelio Hidalgo, Jose Berenguer, Gottfried J. Palm, Winfried Hinrichs, and Uwe T. Bornscheuer. "Cloning, functional expression, biochemical characterization, and structural analysis of a haloalkane dehalogenase from Plesiocystis pacifica SIR-1." Applied Microbiology and Biotechnology 91, no. 4 (May 21, 2011): 1049–60. http://dx.doi.org/10.1007/s00253-011-3328-x.

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Labar, Geoffray, Nathalie Brandt, Amaury Flaba, Johan Wouters, and Laurence Leherte. "Structure and Dynamics of an Archeal Monoglyceride Lipase from Palaeococcus ferrophilus as Revealed by Crystallography and In Silico Analysis." Biomolecules 11, no. 4 (April 3, 2021): 533. http://dx.doi.org/10.3390/biom11040533.

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The crystallographic analysis of a lipase from Palaeococcus ferrophilus (PFL) previously annotated as a lysophospholipase revealed high structural conservation with other monoglyceride lipases, in particular in the lid domain and substrate binding pockets. In agreement with this observation, PFL was shown to be active on various monoacylglycerols. Molecular Dynamics (MD) studies performed in the absence and in the presence of ligands further allowed characterization of the dynamics of this system and led to a systematic closure of the lid compared to the crystal structure. However, the presence of ligands in the acyl-binding pocket stabilizes intermediate conformations compared to the crystal and totally closed structures. Several lid-stabilizing or closure elements were highlighted, i.e., hydrogen bonds between Ser117 and Ile204 or Asn142 and its facing amino acid lid residues, as well as Phe123. Thus, based on this complementary crystallographic and MD approach, we suggest that the crystal structure reported herein represents an open conformation, at least partially, of the PFL, which is likely stabilized by the ligand, and it brings to light several key structural features prone to participate in the closure of the lid.

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Razib, Muhammad Syafiq Mohd, Raja Noor Zaliha Raja Abd Rahman, Fairolniza Mohd Shariff, and Mohd Shukuri Mohamad Ali. "Biochemical and Structural Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Organic Solvent Tolerant Protease." Catalysts 10, no. 1 (January 1, 2020): 55. http://dx.doi.org/10.3390/catal10010055.

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Cross-linked enzyme aggregates (CLEAs) is an immobilization technique that can be used to customize enzymes under an optimized condition. Structural analysis on any enzyme treated with a CLEA remains elusive and has been less explored. In the present work, a method for preparing an organic solvent tolerant protease using a CLEA is disclosed and optimized for better biochemical properties, followed by an analysis of the structure of this CLEA-treated protease. The said organic solvent tolerant protease is a metalloprotease known as elastase strain K in which activity of the metalloprotease is measured by a biochemical interaction with azocasein. Results showed that when a glutaraldehyde of 0.02% (v/v) was used under a 2 h treatment, the amount of recovered activity in CLEA-elastase was highest. The recovered activity of CLEA-elastase and CLEA-elastase-SB (which was a CLEA co-aggregated with starch and bovine serum albumin (BSA)) were at an approximate 60% and 80%, respectively. The CLEA immobilization of elastase strain K allowed the stability of the enzyme to be enhanced at high temperature and at a broader pH. Both CLEA-elastase and CLEA-elastase-SB end-products were able to maintain up to 67% enzyme activity at 60 °C and exhibiting an enhanced stability within pH 5–9 with up to 90% recovering activity. By implementing a CLEA on the organic solvent tolerant protease, the characteristics of the organic solvent tolerant were preserved and enhanced with the presence of 25% (v/v) acetonitrile, ethanol, and benzene at 165%, 173%, and 153% relative activity. Structural analysis through SEM and dynamic light scattering (DLS) showed that CLEA-elastase had a random aggregate morphology with an average diameter of 1497 nm.

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Jendrossek, Dieter, Martina Backhaus, and Meike Andermann. "Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase ofComamonassp. and of its structural gene." Canadian Journal of Microbiology 41, no. 13 (December 15, 1995): 160–69. http://dx.doi.org/10.1139/m95-183.

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The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp. (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium. The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs). This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53 095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards. Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site. By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate. In addition, a conserved region resembling the oxyanion hole of lipases was present. The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins. A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp. depolymerase. The similarities with and differences from other PHA depolymerases are discussed.Key words: biodegradable polymer, poly(3-hydroxybutyrate) depolymerase, serine hydrolase, catalytic triad, Comamonas sp., fibronectin type III modul, substrate-binding site.

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Liu, Lina, Yu Li, Yejing Wang, Peng Zhao, Shuguang Wei, Zhenzhen Li, Huaipu Chang, and Huawei He. "Biochemical characterization and functional analysis of the POU transcription factor POU-M2 of Bombyx mori." International Journal of Biological Macromolecules 86 (May 2016): 701–8. http://dx.doi.org/10.1016/j.ijbiomac.2016.02.016.

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Journal articles on the topic 'Structural analysis and biochemical characterization of lipases'

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