Journal articles on the topic 'N-terminal modification enzymes'
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1
Nakano, Miyako, Sushil K. Mishra, Yuko Tokoro, Keiko Sato, Kazuki Nakajima, Yoshiki Yamaguchi, Naoyuki Taniguchi, and Yasuhiko Kizuka. "Bisecting GlcNAc Is a General Suppressor of Terminal Modification of N-glycan." Molecular & Cellular Proteomics 18, no. 10 (August 2, 2019): 2044–57. http://dx.doi.org/10.1074/mcp.ra119.001534.
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Glycoproteins are decorated with complex glycans for protein functions. However, regulation mechanisms of complex glycan biosynthesis are largely unclear. Here we found that bisecting GlcNAc, a branching sugar residue in N-glycan, suppresses the biosynthesis of various types of terminal epitopes in N-glycans, including fucose, sialic acid and human natural killer-1. Expression of these epitopes in N-glycan was elevated in mice lacking the biosynthetic enzyme of bisecting GlcNAc, GnT-III, and was conversely suppressed by GnT-III overexpression in cells. Many glycosyltransferases for N-glycan terminals were revealed to prefer a nonbisected N-glycan as a substrate to its bisected counterpart, whereas no up-regulation of their mRNAs was found. This indicates that the elevated expression of the terminal N-glycan epitopes in GnT-III-deficient mice is attributed to the substrate specificity of the biosynthetic enzymes. Molecular dynamics simulations further confirmed that nonbisected glycans were preferentially accepted by those glycosyltransferases. These findings unveil a new regulation mechanism of protein N-glycosylation.
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Sakato-Antoku, Miho, Jeremy L. Balsbaugh, and Stephen M. King. "N-Terminal Processing and Modification of Ciliary Dyneins." Cells 12, no. 20 (October 20, 2023): 2492. http://dx.doi.org/10.3390/cells12202492.
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Axonemal dyneins are highly complex microtubule motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At least nineteen cytosolic factors are specifically needed to generate dynein holoenzymes and/or for their trafficking to the growing cilium. Many proteins are subject to N-terminal processing and acetylation, which can generate degrons subject to the AcN-end rule, alter N-terminal electrostatics, generate new binding interfaces, and affect subunit stoichiometry through targeted degradation. Here, we have used mass spectrometry of cilia samples and electrophoretically purified dynein heavy chains from Chlamydomonas to define their N-terminal processing; we also detail the N-terminal acetylase complexes present in this organism. We identify four classes of dynein heavy chain based on their processing pathways by two distinct acetylases, one of which is dependent on methionine aminopeptidase activity. In addition, we find that one component of both the outer dynein arm intermediate/light chain subcomplex and the docking complex is processed to yield an unmodified Pro residue, which may provide a setpoint to direct the cytosolic stoichiometry of other dynein complex subunits that contain N-terminal degrons. Thus, we identify and describe an additional level of processing and complexity in the pathways leading to axonemal dynein formation in cytoplasm.
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STOUGHTON, Daniel M., Gerardo ZAPATA, Robert PICONE, and Willie F. VANN. "Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase." Biochemical Journal 343, no. 2 (October 8, 1999): 397–402. http://dx.doi.org/10.1042/bj3430397.
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Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 → Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in kcat, Km and Ks for CTP, which implicates Arg-12 in catalysis and substrate binding.
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Roll-Mecak, Antonina, Agnieszka Szyk, and Vasilisa Kormendi. "Microtubule chemical complexity: mechanism of tubulin modification enzymes." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1286. http://dx.doi.org/10.1107/s2053273314087130.
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Tubulin is subject to an abundant and diverse set of post-translational modifications that include phosphorylation, acetylation, poly-glutamylation, poly-glycylation and tyrosination. The highest density and variety of post-translational modifications are found in especially complex microtubule arrays like those of neurons or cilia. Not surprisingly, tubulin modification enzymes have been linked to human diseases including cancers and neurodegenerative disorders. I will present recent data from my lab on the mechanism of action of two tubulin modification enzymes that illustrate two divergent paradigms of tubulin recognition. Tubulin tyrosine ligase (TTL) adds a C-terminal Tyr to the exposed C-terminus of alpha-tubulin as part of a tyrosination/detyrosination cycle present in most eukaryotic cells. We solved the first crystal structure of tubulin tyrosine ligase that revealed how the TTL scaffold supported the expansion of the repertory of tubulin post-translational modification enzymes of the TTL like family that recognize either alpha- or beta-tubulin C-terminal tails. In addition to modifying tubulin, TTL also prevents tubulin from incorporating into microtubules by recognizing a tubulin dimer interface that would otherwise be involved in microtubule lattice interactions. I will also present recent work from my group on the structure and mechanism of action of tubulin acetyltransferase (TAT). TAT acetylates Lys-40 on alpha-tubulin in the microtubule lumen. We solved the 2.7Å structure of TAT bound to its ac-coA substrate as well as the 2.45Å structure of a catalytic inactive TAT mutant that reveals a domain swapped dimer in which the functionally essential N-terminus shows evidence of unprecedented structural plasticity. Implications for catalysis and microtubule stimulation of TAT activity will be discussed.
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Lubelski, Jacek, Wout Overkamp, Leon D. Kluskens, Gert N. Moll, and Oscar P. Kuipers. "Influence of Shifting Positions of Ser, Thr, and Cys Residues in Prenisin on the Efficiency of Modification Reactions and on the Antimicrobial Activities of the Modified Prepeptides." Applied and Environmental Microbiology 74, no. 15 (June 6, 2008): 4680–85. http://dx.doi.org/10.1128/aem.00112-08.
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ABSTRACT Since the recent discovery that the nisin modification and transport machinery can be used to produce and modify peptides unrelated to nisin, specific questions arose concerning the specificity of the modification enzymes involved and the limits of their promiscuity with respect to the dehydration and cyclization processes. The nisin leader peptide has been postulated to fulfill a recognition and binding function required for these modifications. Here, we investigated whether the relative positions of the modifiable residues in the nisin prepeptide, with respect to the leader peptide, could influence the efficiency of their modification. We conducted a systematic study on the insertion of one to four alanines in front of either ring A or ring D to change the “reading frame” of modifiable residues, resulting in altered distance and topology of the modifiable residues relative to the leader. The insertion of N-terminal and hinge-located Ala residues had only a modest influence on the modification efficiency, demonstrating that the “phasing” of these residues relative to the leader peptide is not a critical factor in determining modification. However, in all cases, but especially with the N-terminal insertions, the antimicrobial activities of the fully modified nisin species were decreased.
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Nakonieczna, Joanna, Tadeusz Kaczorowski, Agnieszka Obarska-Kosinska, and Janusz M. Bujnicki. "Functional Analysis of MmeI from Methanol Utilizer Methylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes." Applied and Environmental Microbiology 75, no. 1 (November 7, 2008): 212–23. http://dx.doi.org/10.1128/aem.01322-08.
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ABSTRACT MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5′-TCCRAC-3′ (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N 6-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D70-X9-EXK82, characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N 6-adenine γ-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.
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Kelley, M., and D. A. Vessey. "Structural comparison between the mitochondrial aralkyl-CoA and arylacetyl-CoA N-acyltransferases." Biochemical Journal 288, no. 1 (November 15, 1992): 315–17. http://dx.doi.org/10.1042/bj2880315.
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The aralkyl and arylacetyl transferases were purified to homogeneity from bovine kidney by a slight modification of a previous procedure. The M(r) of the arylacetyl transferase was estimated to be 33,500 by SDS/PAGE and that of the aralkyl transferase to be 33,750 by a combination of SDS/PAGE and gel-filtration analysis. N-Terminal-sequence analysis indicated a blocked N-terminus for the arylacetyl transferase and gave the following sequence for the aralkyl transferase: M-F-L-L-Q-G-A-Q-M-L-Q-M-L-E-K. Amino acid analysis revealed differences in composition between the two enzymes. Most notable was the fact that the aralkyl transferase had more methionine and leucine. This difference could be partially accounted for by assuming that the methionine-and-leucine-rich N-terminus was missing from the arylacetyl transferase. Chemical cleavage of the two enzymes at methionine residues using CNBr gave rise to several peptides for each enzyme. N-Terminal-sequence analysis of the 8000-M(r) peptide from the arylacetyl transferase gave a sequence with 69% similarity to the 9000-M(r) peptide from the aralkyl transferase. This was taken to indicate a common origin for the two enzymes.
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Smith, Clyde A., Marta Toth, Nichole K. Stewart, Lauren Maltz, and Sergei B. Vakulenko. "Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2′′-phosphotransferases." Acta Crystallographica Section D Structural Biology 75, no. 12 (November 29, 2019): 1129–37. http://dx.doi.org/10.1107/s2059798319015079.
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Aminoglycoside phosphotransferases (APHs) are one of three families of aminoglycoside-modifying enzymes that confer high-level resistance to the aminoglycoside antibiotics via enzymatic modification. This has now rendered many clinically important drugs almost obsolete. The APHs specifically phosphorylate hydroxyl groups on the aminoglycosides using a nucleotide triphosphate as the phosphate donor. The APH(2′′) family comprises four distinct members, isolated primarily from Enterococcus sp., which vary in their substrate specificities and also in their preference for the phosphate donor (ATP or GTP). The structure of the ternary complex of APH(2′′)-IIIa with GDP and kanamycin was solved at 1.34 Å resolution and was compared with substrate-bound structures of APH(2′′)-Ia, APH(2′′)-IIa and APH(2′′)-IVa. In contrast to the case for APH(2′′)-Ia, where it was proposed that the enzyme-mediated hydrolysis of GTP is regulated by conformational changes in its N-terminal domain upon GTP binding, APH(2′′)-IIa, APH(2′′)-IIIa and APH(2′′)-IVa show no such regulatory mechanism, primarily owing to structural differences in the N-terminal domains of these enzymes.
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Caillava, Celine, Jean Sevalle, and Frederic Checler. "P2-219: identification of enzymes involved in n-terminal truncation and modification of amyloid peptide." Alzheimer's & Dementia 7 (July 2011): S382. http://dx.doi.org/10.1016/j.jalz.2011.05.1101.
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Nashed, Salomé, Houssam El Barbry, Médine Benchouaia, Angélie Dijoux-Maréchal, Thierry Delaveau, Nadia Ruiz-Gutierrez, Lucie Gaulier, et al. "Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import." PLOS Genetics 19, no. 8 (August 16, 2023): e1010848. http://dx.doi.org/10.1371/journal.pgen.1010848.
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N-terminal ends of polypeptides are critical for the selective co-translational recruitment of N-terminal modification enzymes. However, it is unknown whether specific N-terminal signatures differentially regulate protein fate according to their cellular functions. In this work, we developed an in-silico approach to detect functional preferences in cellular N-terminomes, and identified in S. cerevisiae more than 200 Gene Ontology terms with specific N-terminal signatures. In particular, we discovered that Mitochondrial Targeting Sequences (MTS) show a strong and specific over-representation at position 2 of hydrophobic residues known to define potential substrates of the N-terminal acetyltransferase NatC. We validated mitochondrial precursors as co-translational targets of NatC by selective purification of translating ribosomes, and found that their N-terminal signature is conserved in Saccharomycotina yeasts. Finally, systematic mutagenesis of the position 2 in a prototypal yeast mitochondrial protein confirmed its critical role in mitochondrial protein import. Our work highlights the hydrophobicity of MTS N-terminal residues and their targeting by NatC as important features for the definition of the mitochondrial proteome, providing a molecular explanation for mitochondrial defects observed in yeast or human NatC-depleted cells. Functional mapping of N-terminal residues thus has the potential to support the discovery of novel mechanisms of protein regulation or targeting.
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Jayalath, Kumudie, Sean Frisbie, Minhchau To, and Sanjaya Abeysirigunawardena. "Pseudouridine Synthase RsuA Captures an Assembly Intermediate That Is Stabilized by Ribosomal Protein S17." Biomolecules 10, no. 6 (May 30, 2020): 841. http://dx.doi.org/10.3390/biom10060841.
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The ribosome is a large ribonucleoprotein complex that synthesizes protein in all living organisms. Ribosome biogenesis is a complex process that requires synchronization of various cellular events, including ribosomal RNA (rRNA) transcription, ribosome assembly, and processing and post-transcriptional modification of rRNA. Ribosome biogenesis is fine-tuned with various assembly factors, possibly including nucleotide modification enzymes. Ribosomal small subunit pseudouridine synthase A (RsuA) pseudouridylates U516 of 16S helix 18. Protein RsuA is a multi-domain protein that contains the N-terminal peripheral domain, which is structurally similar to the ribosomal protein S4. Our study shows RsuA preferably binds and pseudouridylates an assembly intermediate that is stabilized by ribosomal protein S17 over the native-like complex. In addition, the N-terminal domain truncated RsuA showed that the presence of the S4-like domain is important for RsuA substrate recognition.
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Lorenz, Sonja. "Structural mechanisms of HECT-type ubiquitin ligases." Biological Chemistry 399, no. 2 (January 26, 2018): 127–45. http://dx.doi.org/10.1515/hsz-2017-0184.
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AbstractUbiquitin ligases (E3 enzymes) transfer ubiquitin from ubiquitin-conjugating (E2) enzymes to target proteins. By determining the selection of target proteins, modification sites on those target proteins, and the types of ubiquitin modifications that are formed, E3 enzymes are key specificity factors in ubiquitin signaling. Here, I summarize our knowledge of the structural mechanisms in the HECT E3 subfamily, many members of which play important roles in human disease. I discuss interactions of the conserved HECT domain with E2 enzymes, ubiquitin and target proteins, as well as macromolecular interactions with regulatory functions. While we understand individual steps in the catalytic cycle of HECT E3 enzymes on a structural level, this review also highlights key aspects that have yet to be elucidated. For instance, it remains unclear how diverse target proteins are presented to the catalytic center and how certain HECT E3 enzymes achieve specificity in ubiquitin linkage formation. The structural and functional properties of the N-terminal regions of HECT E3 enzymes that likely act as signaling hubs are also largely unknown. Structural insights into these aspects may open up routes for a therapeutic intervention with specific HECT E3 functions in distinct pathophysiological settings.
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Mann, Sarah A., Megan K. DeMart, Braidy May, Corey P. Causey, and Bryan Knuckley. "Histone H4-based peptoids are inhibitors of protein arginine methyltransferase 1 (PRMT1)." Biochemical Journal 477, no. 16 (August 21, 2020): 2971–80. http://dx.doi.org/10.1042/bcj20200534.
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Methylation of arginine residues occurs on a number of protein substrates, most notably the N-terminal tails of histones, and is catalyzed by a family of enzymes called the protein arginine methyltransferases (PRMTs). This modification can lead to transcriptional activation or repression of cancer-related genes. To date, a number of inhibitors, based on natural peptide substrates, have been developed for the PRMT family of enzymes. However, because peptides are easily degraded in vivo, the utility of these inhibitors as potential therapeutics is limited. The use of peptoids, which are peptide mimetics where the amino acid side chain is attached to the nitrogen in the amide backbone instead of the α-carbon, may circumvent the problems associated with peptide degradation. Given the structural similarities, peptoid scaffolds may provide enhanced stability, while preserving the mechanism of action. Herein, we have identified that peptoids based on natural peptide substrates are not catalyzed to the product by PRMT1, but instead are inhibitors of this enzyme. Reducing the length of the peptoid reduces inhibition and suggest the residues distal from the site of modification are important for binding. Furthermore, a positive charge on the N-terminus helps promote binding and improves inhibition. Selectivity among family members is likely possible based on inhibition being moderately selective for PRMT1 over PRMT5 and provides a scaffold that can be used to develop pharmaceuticals against this class of enzymes.
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Hayman, A. R., A. J. Dryden, T. J. Chambers, and M. J. Warburton. "Tartrate-resistant acid phosphatase from human osteoclastomas is translated as a single polypeptide." Biochemical Journal 277, no. 3 (August 1, 1991): 631–34. http://dx.doi.org/10.1042/bj2770631.
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Tartrate-resistant acid phosphatases have been isolated from a number of sources. These enzymes consist of one subunit (Mr 30,000-40,000) or two dissimilar subunits (Mr 15,000-20,000). Previously we isolated the enzyme from human osteoclastomas, as a two-subunit protein. By Northern blotting and hybridization with radiolabelled oligonucleotides corresponding to the N-terminal sequences of the two subunits, we demonstrate here that the enzyme is transcribed as one mRNA which is translated in vitro to produce a single polypeptide of approx. Mr 33,000. Transcription as a single mRNA species is also the case in other tissues. These results suggest that the osteoclastoma enzyme undergoes post-translational modification in the form of cleavage of a single peptide bond to give a disulphide-bonded two-subunit protein.
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Jones, Carys S., David Sychantha, P. Lynne Howell, and Anthony J. Clarke. "Structural basis for the O-acetyltransferase function of the extracytoplasmic domain of OatA from Staphylococcus aureus." Journal of Biological Chemistry 295, no. 24 (April 29, 2020): 8204–13. http://dx.doi.org/10.1074/jbc.ra120.013108.
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Many bacteria possess enzymes that modify the essential cell-wall polymer peptidoglycan by O-acetylation. This modification occurs in numerous Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, a common cause of human infections. O-Acetylation of peptidoglycan protects bacteria from the lytic activity of lysozyme, a mammalian innate immune enzyme, and as such is important for bacterial virulence. The O-acetylating enzyme in Gram-positive bacteria, O-acetyltransferase A (OatA), is a two-domain protein consisting of an N-terminal integral membrane domain and a C-terminal extracytoplasmic domain. Here, we present the X-ray crystal structure at 1.71 Å resolution and the biochemical characterization of the C-terminal domain of S. aureus OatA. The structure revealed that this OatA domain adopts an SGNH-hydrolase fold and possesses a canonical catalytic triad. Site-specific replacement of active-site amino acids revealed the presence of a water-coordinating aspartate residue that limits esterase activity. This residue, although conserved in staphyloccocal OatA and most other homologs, is not present in the previously characterized streptococcal OatA. These results provide insights into the mechanism of acetyl transfer in the SGNH/GDSL hydrolase family and highlight important evolutionary differences between homologous OatA enzymes. Furthermore, this study enhances our understanding of PG O-acetyltransferases, which could guide the development of novel antibacterial drugs to combat infections with multidrug-resistant bacterial pathogens.
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Nosrati, Meisam, Debayan Dey, Atousa Mehrani, Sarah E. Strassler, Natalia Zelinskaya, Eric D. Hoffer, Scott M. Stagg, Christine M. Dunham, and Graeme L. Conn. "Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition." Journal of Biological Chemistry 294, no. 46 (October 8, 2019): 17642–53. http://dx.doi.org/10.1074/jbc.ra119.011181.
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Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m1A1408) methyltransferases, suggesting that both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification but did not directly contribute to the binding affinity. The results from our experiments define the critical features of m7G1405 methyltransferase–substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.
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Kolli, Nagamalleswari, Jowita Mikolajczyk, Marcin Drag, Debaditya Mukhopadhyay, Nela Moffatt, Mary Dasso, Guy Salvesen, and Keith D. Wilkinson. "Distribution and paralogue specificity of mammalian deSUMOylating enzymes." Biochemical Journal 430, no. 2 (August 13, 2010): 335–44. http://dx.doi.org/10.1042/bj20100504.
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The covalent attachment of SUMO (small ubiquitin-like protein modifier) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalysed by SENPs (SUMO-specific processing proteases). Mammals contain four SUMO paralogues and six SENP enzymes. In the present paper, we describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal cSENPs (SENP catalytic domains). We first characterized the reaction of each endogenous SENP and cSENPs with HA–SUMO-VS [HA (haemagglutinin)-tagged SUMO-vinyl sulfones], active-site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogues, whereas all other endogeneous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1- or SUMO2-AMC (7-amino-4-methylcoumarin) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by Km and kcat, whereas cSENP7 works only on SUMO2. Compared with cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity, indicating that paralogue specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggest that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.
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Juntawong, Piyada, Pimprapai Butsayawarapat, Pattralak Songserm, Ratchaneeporn Pimjan, and Supachai Vuttipongchaikij. "Overexpression of Jatropha curcas ERFVII2 Transcription Factor Confers Low Oxygen Tolerance in Transgenic Arabidopsis by Modulating Expression of Metabolic Enzymes and Multiple Stress-Responsive Genes." Plants 9, no. 9 (August 20, 2020): 1068. http://dx.doi.org/10.3390/plants9091068.
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Enhancing crop tolerance to waterlogging is critical for improving food and biofuel security. In waterlogged soils, roots are exposed to a low oxygen environment. The group VII ethylene response factors (ERFVIIs) were recently identified as key regulators of plant low oxygen response. Oxygen-dependent N-end rule pathways can regulate the stability of ERFVIIs. This study aims to characterize the function of the Jatropha curcas ERFVIIs and the impact of N-terminal modification that stabilized the protein toward low oxygen response. This study revealed that all three JcERFVII proteins are substrates of the N-end rule pathway. Overexpression of JcERFVII2 conferred tolerance to low oxygen stress in Arabidopsis. In contrast, the constitutive overexpression of stabilized JcERFVII2 reduced low oxygen tolerance. RNA-seq was performed to elucidate the functional roles of JcERFVII2 and the impact of its N-terminal modification. Overexpression of both wildtype and stabilized JcERFVII2 constitutively upregulated the plant core hypoxia-responsive genes. Besides, overexpression of the stabilized JcERFVII2 further upregulated various genes controlling fermentative metabolic processes, oxidative stress, and pathogen responses under aerobic conditions. In summary, JcERFVII2 is an N-end rule regulated waterlogging-responsive transcription factor that modulates the expression of multiple stress-responsive genes; therefore, it is a potential candidate for molecular breeding of multiple stress-tolerant crops.
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Galati, Rossella, Alessandra Verdina, Giuliana Falasca, and Alberto Chersi. "Increased Resistance of Peptides to Serum Proteases by Modification of their Amino Groups." Zeitschrift für Naturforschung C 58, no. 7-8 (August 1, 2003): 558–61. http://dx.doi.org/10.1515/znc-2003-7-819.
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Abstract The ability of synthetic protein fragments to survive the degradative action of aminopeptidases and serum proteolytic enzymes can be remarkably enhanced by slight modifications at their N-terminal alpha-amino group. This can be achieved by addition of beta-alanine or amino acids of the d-configuration, amino acids which are seldom found in a living organism. These modifications do scarcely modify the chemical and physical properties of the peptides, and should be preferrred, especially for in vivo tests, to drastic alterations of peptides as produced by dinitrophenylation or dansylation of the amino groups.
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Ben-Arie, N., M. Khen, and D. Lancet. "Glutathione S-transferases in rat olfactory epithelium: purification, molecular properties and odorant biotransformation." Biochemical Journal 292, no. 2 (June 1, 1993): 379–84. http://dx.doi.org/10.1042/bj2920379.
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The olfactory epithelium is exposed to a variety of xenobiotic chemicals, including odorants and airborne toxic compounds. Recently, two novel, highly abundant, olfactory-specific biotransformation enzymes have been identified: cytochrome P-450olf1 and olfactory UDP-glucuronosyltransferase (UGT(olf)). The latter is a phase II biotransformation enzyme which catalyses the glucuronidation of alcohols, thiols, amines and carboxylic acids. Such covalent modification, which markedly affects lipid solubility and agonist potency, may be particularly important in the rapid termination of odorant signals. We report here the identification and characterization of a second olfactory phase II biotransformation enzyme, a glutathione S-transferase (GST). The olfactory epithelial cytosol shows the highest GST activity among the extrahepatic tissues examined. Significantly, olfactory epithelium had an activity 4-7 times higher than in other airway tissues, suggesting a role for this enzyme in chemoreception. The olfactory GST has been affinity-purified to homogeneity, and shown by h.p.l.c. and N-terminal amino acid sequencing to constitute mainly the Yb1 and Yb2 subunits, different from most other tissues that have mixtures of more enzyme classes. The identity of the olfactory enzymes was confirmed by PCR cloning and restriction enzyme analysis. Most importantly, the olfactory GSTs were found to catalyse glutathione conjugation of several odorant classes, including many unsaturated aldehydes and ketones, as well as epoxides. Together with UGT(olf), olfactory GST provides the necessary broad coverage of covalent modification capacity, which may be crucial for the acuity of the olfactory process.
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Millar, A. Harvey, Joshua L. Heazlewood, Carmela Giglione, Michael J. Holdsworth, Andreas Bachmair, and Waltraud X. Schulze. "The Scope, Functions, and Dynamics of Posttranslational Protein Modifications." Annual Review of Plant Biology 70, no. 1 (April 29, 2019): 119–51. http://dx.doi.org/10.1146/annurev-arplant-050718-100211.
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Assessing posttranslational modification (PTM) patterns within protein molecules and reading their functional implications present grand challenges for plant biology. We combine four perspectives on PTMs and their roles by considering five classes of PTMs as examples of the broader context of PTMs. These include modifications of the N terminus, glycosylation, phosphorylation, oxidation, and N-terminal and protein modifiers linked to protein degradation. We consider the spatial distribution of PTMs, the subcellular distribution of modifying enzymes, and their targets throughout the cell, and we outline the complexity of compartmentation in understanding of PTM function. We also consider PTMs temporally in the context of the lifetime of a protein molecule and the need for different PTMs for assembly, localization, function, and degradation. Finally, we consider the combined action of PTMs on the same proteins, their interactions, and the challenge ahead of integrating PTMs into an understanding of protein function in plants.
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Su, Ping, Heejeong Im, Hsiaoling Hsieh, Simon Kang’A, and Noel W. Dunn. "LlaFI, a Type III Restriction and Modification System in Lactococcus lactis." Applied and Environmental Microbiology 65, no. 2 (February 1, 1999): 686–93. http://dx.doi.org/10.1128/aem.65.2.686-693.1999.
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ABSTRACT We describe a type III restriction and modification (R/M) system,LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactisLL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence.LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.
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Davis, Katherine M., Kelsey R. Schramma, William A. Hansen, John P. Bacik, Sagar D. Khare, Mohammad R. Seyedsayamdost, and Nozomi Ando. "Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition." Proceedings of the National Academy of Sciences 114, no. 39 (September 11, 2017): 10420–25. http://dx.doi.org/10.1073/pnas.1703663114.
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Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine–tryptophan cross-link, which is installed by the radical S-adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon–carbon cross-link in the streptide family of natural products.
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Plavner, Noa, and Jerry Eichler. "Defining the Topology of the N-Glycosylation Pathway in the Halophilic Archaeon Haloferax volcanii." Journal of Bacteriology 190, no. 24 (October 17, 2008): 8045–52. http://dx.doi.org/10.1128/jb.01200-08.
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ABSTRACT In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the H. volcanii S-layer glycoprotein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the H. volcanii S-layer glycoprotein as occurring within the cell.
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Chumpen Ramirez, Sabrina, Fernando M. Ruggiero, Jose Luis Daniotti, and Javier Valdez Taubas. "Ganglioside glycosyltransferases are S-acylated at conserved cysteine residues involved in homodimerisation." Biochemical Journal 474, no. 16 (August 7, 2017): 2803–16. http://dx.doi.org/10.1042/bcj20170124.
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Ganglioside glycosyltransferases (GGTs) are type II membrane proteins bearing a short N-terminal cytoplasmic tail, a transmembrane domain (TMD), and a lumenal catalytic domain. The expression and activity of these enzymes largely determine the quality of the glycolipids that decorate mammalian cell membranes. Many glycosyltransferases (GTs) are themselves glycosylated, and this is important for their proper localisation, but few if any other post-translational modifications of these proteins have been reported. Here, we show that the GGTs, ST3Gal-V, ST8Sia-I, and β4GalNAcT-I are S-acylated at conserved cysteine residues located close to the cytoplasmic border of their TMDs. ST3Gal-II, a GT that sialylates glycolipids and glycoproteins, is also S-acylated at a conserved cysteine located in the N-terminal cytoplasmic tail. Many other GTs also possess cysteine residues in their cytoplasmic regions, suggesting that this modification occurs also on these GTs. S-acylation, commonly known as palmitoylation, is catalysed by a family of palmitoyltransferases (PATs) that are mostly localised at the Golgi complex but also at the endoplasmic reticulum (ER) and the plasma membrane. Using GT ER retention mutants, we found that S-acylation of β4GalNAcT-I and ST3Gal-II takes place at different compartments, suggesting that these enzymes are not substrates of the same PAT. Finally, we found that cysteines that are the target of S-acylation on β4GalNAcT-I and ST3Gal-II are involved in the formation of homodimers through disulphide bonds. We observed an increase in ST3Gal-II dimers in the presence of the PAT inhibitor 2-bromopalmitate, suggesting that GT homodimerisation may be regulating S-acylation
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LI, Lin, Song LING, Chun-lai WU, Wei-zhe YAO, and Gen-jun XU. "Separate bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: the role of the C-terminal tail in modulating enzyme activity." Biochemical Journal 328, no. 3 (December 15, 1997): 751–56. http://dx.doi.org/10.1042/bj3280751.
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The separate bisphosphatase domain (amino acid residues 243-468) of the chicken liver bifunctional enzyme 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase was expressed in Escherichia coli and purified to homogeneity. The fructose-2,6-bisphosphatase activity of the separate domain was 7-fold higher than that of the native bifunctional enzyme, and exhibited substrate inhibition characteristic of the native enzyme. The inhibition of the enzymes by fructose 2,6-bisphosphate could be overcome by Pi, glycerol 3-phosphate and GTP. Deletion of 30 amino acid residues from the C-terminus of the separate domain resulted in around a 5-fold increase in the Vmax of the bisphosphatase. Also, the truncated form was more accessible to chemical modification by diethyl pyrocarbonate and N-ethylmaleimide, suggesting a more open structure than the wild-type form. In addition, the mutation of cysteine-389 to alanine increased bisphosphatase activity by 20% and the Km value for fructose 2,6-bisphosphate by 3-fold, and both the point mutation at cysteine-389 and the deletional mutation led to the predominantly insoluble expression of the enzyme. The results indicated that the C-terminal tail plays a role in modulating the enzyme activity and suggested that the difference in the C-terminal tail sequence is responsible for the difference in activity of the chicken and rat liver fructose-2,6-bisphosphatases. It is postulated that an interaction between the C-terminal tail and the active site might be present.
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Raz, A., and P. Needleman. "Differential modification of cyclo-oxygenase and peroxidase activities of prostaglandin endoperoxidase synthase by proteolytic digestion and hydroperoxides." Biochemical Journal 269, no. 3 (August 1, 1990): 603–7. http://dx.doi.org/10.1042/bj2690603.
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Prostaglandin endoperoxide synthase (PES, EC 1.14.99.1) catalyse the conversion of arachidonic acid into prostaglandin H2. The enzyme is a 140 kDa homodimer which contains both a cyclo-oxygenase activity (converting arachidonate into prostaglandin G2) and peroxidase activity (reducing prostaglandin G2 to H2). PES undergoes rapid self-inactivation during oxygenation of arachidonate to prostaglandin H2 in vitro. The previously reported cDNA-derived amino acid sequence indicates numerous sites for trypsin or thrombin cleavage. Most of these sites must be inaccessible, since these enzymes cleave only at Arg253. The enzyme appears to be a self-adherent and highly folded molecule, since after cleavage it retains its functional assembly and its homodimer size of 140 kDa, as well as its overall enzymic activity. Only under denaturing conditions (e.g. SDS/PAGE) can the proteolytic peptides be demonstrated: a 38 kDa C-terminal fragment containing the aspirin-derived-acetyl-binding ability, and a 33 kDa N-terminal fragment. In the present studies we investigated whether the two enzymic activities of PES can be differentially manipulated by proteolytic cleavage or by substrate (arachidonate) self-inactivation. The results indicated that, during arachidonate oxygenation by PES, the cyclooxygenase activity is selectively inactivated, whereas the peroxidase activity is essentially retained. By contrast, thrombin or trypsin cleavage of pure PES or microsomal PES (to yield the 38 and 33 kDa peptide fragments) inactivated the peroxidase, but not the cyclo-oxygenase. Taken together, these results suggest the presence of separate cyclo-oxygenase and peroxidase structural domains on the enzyme.
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FOLEY, Vivienne, and David SHEEHAN. "Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass." Biochemical Journal 333, no. 3 (August 1, 1998): 839–45. http://dx.doi.org/10.1042/bj3330839.
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Two similar glutathione S-transferases (GSTs), which do not bind to glutathione– or S-hexylglutathione–agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243–248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.
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Peng, Gui-Xin, Yong Zhang, Qin-Qin Wang, Qing-Run Li, Hong Xu, En-Duo Wang, and Xiao-Long Zhou. "The human tRNA taurine modification enzyme GTPBP3 is an active GTPase linked to mitochondrial diseases." Nucleic Acids Research 49, no. 5 (February 22, 2021): 2816–34. http://dx.doi.org/10.1093/nar/gkab104.
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Abstract GTPBP3 and MTO1 cooperatively catalyze 5-taurinomethyluridine (τm5U) biosynthesis at the 34th wobble position of mitochondrial tRNAs. Mutations in tRNAs, GTPBP3 or MTO1, causing τm5U hypomodification, lead to various diseases. However, efficient in vitro reconstitution and mechanistic study of τm5U modification have been challenging, in part due to the lack of pure and active enzymes. A previous study reported that purified human GTPBP3 (hGTPBP3) is inactive in GTP hydrolysis. Here, we identified the mature form of hGTPBP3 and showed that hGTPBP3 is an active GTPase in vitro that is critical for tRNA modification in vivo. Unexpectedly, the isolated G domain and a mutant with the N-terminal domain truncated catalyzed GTP hydrolysis to only a limited extent, exhibiting high Km values compared with that of the mature enzyme. We further described several important pathogenic mutations of hGTPBP3, associated with alterations in hGTPBP3 localization, structure and/or function in vitro and in vivo. Moreover, we discovered a novel cytoplasm-localized isoform of hGTPBP3, indicating an unknown potential noncanonical function of hGTPBP3. Together, our findings established, for the first time, the GTP hydrolysis mechanism of hGTPBP3 and laid a solid foundation for clarifying the τm5U modification mechanism and etiology of τm5U deficiency-related diseases.
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Raj, Hanumantharao G., Ranju Kumari, Garima Gupta, Rajesh Kumar, Daman Saluja, Kambadoor M. Muralidhar, Ajit Kumar, et al. "Novel function of calreticulin: Characterization of calreticulin as a transacetylase-mediating protein acetylator independent of acetyl CoA using polyphenolic acetates." Pure and Applied Chemistry 78, no. 5 (January 1, 2006): 985–92. http://dx.doi.org/10.1351/pac200678050985.
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Our earlier investigations culminated in the discovery of a unique membrane-bound enzyme in mammalian cells catalyzing the transfer of acetyl group from polyphenolic acetates (PAs) to certain functional proteins, resulting in the modulation of their activities. This enzyme was termed acetoxy drug:protein transacetylase (TAase) since it acted upon several classes of PAs. TAase was purified from rat liver microsomes to homogeneity and exhibited the molecular weight of 55 KDa. TAase-catalyzed protein acetylation by PAs was evidenced by the demonstration of immunoreactivity of the acetylated target protein such as nitric oxide synthase (NOS) with anti-acetyl lysine. The possible acetylation of human platelet NOS by PA as described above resulted in the enhancement of intracellular levels of nitric oxide (NO). PAs unlike the parent polyphenols were found to exhibit NO-related physiological effects. The N-terminal sequence was found to show 100 % homology with N-terminal sequence of mature calreticulin (CRT). The identity of TAase with CRT, an endoplasmic reticulum (ER) protein, was evidenced by the demonstration of the properties of CRT such as immunoreactivity with anti-calreticulin, binding to Ca2+ ions and being substrate for phosphorylation by protein kinase c (PKC), which are the hallmark characteristics of CRT. These observations for the first time convincingly attribute the transacetylase function to CRT, which possibly plays an important role in protein modification by way of carrying out acetylation of various enzymes through a biochemical mechanism independent of acetyl CoA.
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MOORE, Allison B., and Sheldon W. MAY. "Kinetic and inhibition studies on substrate channelling in the bifunctional enzyme catalysing C-terminal amidation." Biochemical Journal 341, no. 1 (June 24, 1999): 33–40. http://dx.doi.org/10.1042/bj3410033.
Full textAbstract:
A series of experiments has been conducted to investigate the possibility that substrate channelling might occur in the bifunctional forms of enzymes carrying out C-terminal amidation, a post-translational modification essential to the biological activity of many neuropeptides. C-terminal amidation entails sequential action by peptidylglycine mono-oxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5), with the mono-oxygenase catalysing conversion of a glycine-extended pro-peptide into the corresponding α-hydroxyglycine derivative, which is then converted by the lyase into amidated peptide plus glyoxylate. Since the mono-oxygenase and lyase reactions exhibit tandem reaction stereospecificities, channelling of the α-hydroxy intermediate might occur, as is the case for some other multifunctional enzymes. Selective inhibition of the mono-oxygenase domain by competitive ester inhibitors, as well as mechanism-based mono-oxygenase inactivation by the novel olefinic inhibitor 5-acetamido-4-oxo-6-phenylhex-2-enoate (N-acetylphenylalanyl acrylate), has little to no effect on the kinetic parameters of the lyase domain of the AE from Xenopus laevis. Similarly, inhibition of the lyase domain by the potent dioxo inhibitor 2,4-dioxo-5-acetamido-6-phenylhexanoate has little effect on the activity of the monooxygenase domain in the bifunctional enzyme. A series of experiments on intermediate accumulation and conversion were also carried out, along with kinetic investigations of the reactivities of the monofunctional and bifunctional forms of PAM and PGL towards substrates and inhibitors. Taken together, the results demonstrate the kinetic independence of the mono-oxygenase and lyase domains, and provide no evidence for substrate channelling between these domains in the bifunctional amidating enzyme.
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Zhang, Yaoping, Edward L. Pohlmann, Jose Serate, Mary C. Conrad, and Gary P. Roberts. "Mutagenesis and Functional Characterization of the Four Domains of GlnD, a Bifunctional Nitrogen Sensor Protein." Journal of Bacteriology 192, no. 11 (April 2, 2010): 2711–21. http://dx.doi.org/10.1128/jb.01674-09.
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ABSTRACT GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of PII protein; PII in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants.
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Lidholt, K., and U. Lindahl. "Biosynthesis of heparin. The d-glucuronosyl- and N-acetyl-d-glucosaminyltransferase reactions and their relation to polymer modification." Biochemical Journal 287, no. 1 (October 1, 1992): 21–29. http://dx.doi.org/10.1042/bj2870021.
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Oligosaccharides with the general structure [GlcA-GlcNAc]n-GlcA-aMan (aMan is 2,5-anhydro-D-mannose), derived from the Escherichia coli K5 capsular polysaccharide, were found to serve as monosaccharide acceptors for a GlcNAc-transferase, solubilized from a mouse mastocytoma microsomal fraction and implicated in heparin biosynthesis. Digestion of these oligosaccharides with beta-D-glucuronidase yielded acceptors for the GlcA-transferase that acts in concert with the GlcNAc-transferase. Assays based on the oligosaccharide acceptors showed broad pH optima for the two enzymes, centred around pH 6.5 for the GlcNAc-transferase and around pH 7.0 for the GlcA-transferase. The GlcNAc-transferase showed an absolute requirement for Mn2+, whereas the GlcA-transferase was stimulated by Ca2+ and Mg2+ but not by Mn2+. The GlcNAc acceptor ability of the [GlcA-GlcNAc]n-GlcA-aMan oligosaccharides increased with increasing chain length, as reflected by the apparent Km, which was 60 microM for a hexasaccharide but 6 microM for a hexadecasaccharide. By contrast, the Km for [GlcNAc-GlcA]n-aMan oligosaccharides in the GlcA-transferase reaction was higher, approximately 0.5 mM, and unaffected by acceptor size. After chemical modification of the oligosaccharides to obtain mixed N-substituents (N-unsubstituted, N-acetylated or N-sulphated GlcN residues), GlcNAc transfer was found to be virtually independent of the N-substituent pattern of the acceptor sequence. The GlcA-transferase, on the other hand, showed marked preference for an acceptor with a non-reducing-terminal GlcNAc-GlcA-GlcNSO3- sequence, which would thus have a lower Km for the enzyme than the corresponding fully N-acetylated structure. These results, along with our previous finding that chain elongation in a mastocytoma microsomal system is strongly promoted by concomitant N-sulphation of the nascent chain [Lidholt, Kjellén & Lindahl (1989) Biochem. J. 261, 999-1007], raise the possibility that the glycosyltransferases and the N-deacetylase/N-sulphotransferase act in concert during chain elongation, assembled into an enzyme complex.
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São-José, Carlos, Ricardo Parreira, Graça Vieira, and Mário A. Santos. "The N-Terminal Region of the Oenococcus oeniBacteriophage fOg44 Lysin Behaves as a Bona Fide Signal Peptide inEscherichia coli and as a cis-Inhibitory Element, Preventing Lytic Activity on Oenococcal Cells." Journal of Bacteriology 182, no. 20 (October 15, 2000): 5823–31. http://dx.doi.org/10.1128/jb.182.20.5823-5831.2000.
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ABSTRACT The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed inE. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeniand Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.
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Goris, Marianne, Robert S. Magin, Håvard Foyn, Line M. Myklebust, Sylvia Varland, Rasmus Ree, Adrian Drazic, et al. "Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80." Proceedings of the National Academy of Sciences 115, no. 17 (March 26, 2018): 4405–10. http://dx.doi.org/10.1073/pnas.1719251115.
Full textAbstract:
N-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation. In this work, we developed potent and selective bisubstrate inhibitors for NAA80 and determined the crystal structure of NAA80 in complex with such an inhibitor, revealing that NAA80 adopts a fold similar to other NAT enzymes but with a more open substrate binding region. Furthermore, in contrast to most other NATs, the substrate specificity of NAA80 is mainly derived through interactions between the enzyme and the acidic amino acids at positions 2 and 3 of the actin substrate and not residues 1 and 2. A yeast model revealed that ectopic expression of NAA80 in a strain lacking NatB activity partially restored Nt acetylation of NatB substrates, including yeast actin. Thus, NAA80 holds intrinsic capacity to posttranslationally Nt acetylate NatB-type substrates in vivo. In sum, the presence of a dominant cotranslational NatB in all eukaryotes, the specific posttranslational actin methionine removal in animals, and finally, the unique structural features of NAA80 leave only the processed actins as in vivo substrates of NAA80. Together, this study reveals the molecular and cellular basis of NAA80 Nt acetylation and provides a scaffold for development of inhibitors for the regulation of cytoskeletal properties.
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Shang, Tao, Chee Mun Fang, Chin Eng Ong, and Yan Pan. "Heterologous Expression of Recombinant Human Cytochrome P450 (CYP) in Escherichia coli: N-Terminal Modification, Expression, Isolation, Purification, and Reconstitution." BioTech 12, no. 1 (February 7, 2023): 17. http://dx.doi.org/10.3390/biotech12010017.
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Cytochrome P450 (CYP) enzymes play important roles in metabolising endogenous and xenobiotic substances. Characterisations of human CYP proteins have been advanced with the rapid development of molecular technology that allows heterologous expression of human CYPs. Among several hosts, bacteria systems such as Escherichia coli (E. coli) have been widely used thanks to their ease of use, high level of protein yields, and affordable maintenance costs. However, the levels of expression in E. coli reported in the literature sometimes differ significantly. This paper aims to review several contributing factors, including N-terminal modifications, co-expression with a chaperon, selections of vectors and E. coli strains, bacteria culture and protein expression conditions, bacteria membrane preparations, CYP protein solubilizations, CYP protein purifications, and reconstitution of CYP catalytic systems. The common factors that would most likely lead to high expression of CYPs were identified and summarised. Nevertheless, each factor may still require careful evaluation for individual CYP isoforms to achieve a maximal expression level and catalytic activity. Recombinant E. coli systems have been evidenced as a useful tool in obtaining the ideal level of human CYP proteins, which ultimately allows for subsequent characterisations of structures and functions.
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Yang, Chien-I., Hao-Hsuan Hsieh, and Shu-ou Shan. "Timing and specificity of cotranslational nascent protein modification in bacteria." Proceedings of the National Academy of Sciences 116, no. 46 (October 30, 2019): 23050–60. http://dx.doi.org/10.1073/pnas.1912264116.
Full textAbstract:
The nascent polypeptide exit site of the ribosome is a crowded environment where multiple ribosome-associated protein biogenesis factors (RPBs) compete for the nascent polypeptide to influence their localization, folding, or quality control. Here we address how N-terminal methionine excision (NME), a ubiquitous process crucial for the maturation of over 50% of the bacterial proteome, occurs in a timely and selective manner in this crowded environment. In bacteria, NME is mediated by 2 essential enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). We show that the reaction of MAP on ribosome-bound nascent chains approaches diffusion-limited rates, allowing immediate methionine excision of optimal substrates after deformylation. Specificity is achieved by kinetic competition of NME with translation elongation and by regulation from other RPBs, which selectively narrow the processing time window for suboptimal substrates. A mathematical model derived from the data accurately predicts cotranslational NME efficiency in the cytosol. Our results demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity, and provides a platform to study other cotranslational protein biogenesis pathways.
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Millar, D. J., A. K. Allen, C. G. Smith, C. Sidebottom, A. R. Slabas, and G. P. Bolwell. "Chitin-binding proteins in potato (Solanum tuberosum L.) tuber. Characterization, immunolocalization and effects of wounding." Biochemical Journal 283, no. 3 (May 1, 1992): 813–21. http://dx.doi.org/10.1042/bj2830813.
Full textAbstract:
Tubers of potato (Solanum tuberosum L.) contain a number of chitin-binding proteins which have possible functions in defence against pathogens. A major protein of the tuber is the chitin-binding lectin which has been further characterized with respect to its antigenicity and N-terminal amino acid sequence. By using an antiserum monospecific for tuber lectin in unwounded potato the protein was found in the cytoplasm and vacuole, unusually for a hydroxyproline-rich glycoprotein, but consistent with its soluble nature in subcellular extracts. Little increased synthesis of the lectin precursor or the post-translationally modified form could be demonstrated in excised potato tuber discs. However, after wounding there is increased synthesis of another hydroxyproline-containing glycoprotein of Mr 57,000, which binds to chitin and shares common epitopes with the lectin. In comparison with the tuber lectin, this novel glycoprotein contains less hydroxyproline, but from its overall composition it is clearly not an underhydroxylated form of the tuber lectin. It differed in its N-terminal amino acid sequence and was much less glycosylated, although arabinose was still present. Synthesis of the Mr-57,000 polypeptide began after the initial burst of protein synthesis and increased, reaching a peak at 24 h after wounding. The protein was produced with its enzymes of post-translational modification, prolyl hydroxylase and arabinosyltransferase, concomitantly with the marker enzymes for wounding, phenylalanine ammonia-lyase and membrane-bound phenol oxidase and peroxidase.
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Scott, Hannah, Gideon J. Davies, and Zachary Armstrong. "The structure of Phocaeicola vulgatus sialic acid acetylesterase." Acta Crystallographica Section D Structural Biology 78, no. 5 (April 26, 2022): 647–57. http://dx.doi.org/10.1107/s2059798322003357.
Full textAbstract:
Sialic acids terminate many N- and O-glycans and are widely distributed on cell surfaces. There are a diverse range of enzymes which interact with these sugars throughout the tree of life. They can act as receptors for influenza and specific betacoronaviruses in viral binding and their cleavage is important in virion release. Sialic acids are also exploited by both commensal and pathogenic bacteria for nutrient acquisition. A common modification of sialic acid is 9-O-acetylation, which can limit the action of sialidases. Some bacteria, including human endosymbionts, employ esterases to overcome this modification. However, few bacterial sialic acid 9-O-acetylesterases (9-O-SAEs) have been structurally characterized. Here, the crystal structure of a 9-O-SAE from Phocaeicola vulgatus (PvSAE) is reported. The structure of PvSAE was determined to resolutions of 1.44 and 2.06 Å using crystals from two different crystallization conditions. Structural characterization revealed PvSAE to be a dimer with an SGNH fold, named after the conserved sequence motif of this family, and a Ser–His–Asp catalytic triad. These structures also reveal flexibility in the most N-terminal α-helix, which provides a barrier to active-site accessibility. Biochemical assays also show that PvSAE deacetylates both mucin and the acetylated chromophore para-nitrophenyl acetate. This structural and biochemical characterization of PvSAE furthers the understanding of 9-O-SAEs and may aid in the discovery of small molecules targeting this class of enzyme.
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Scott, Hannah, Gideon J. Davies, and Zachary Armstrong. "The structure of Phocaeicola vulgatus sialic acid acetylesterase." Acta Crystallographica Section D Structural Biology 78, no. 5 (April 26, 2022): 647–57. http://dx.doi.org/10.1107/s2059798322003357.
Full textAbstract:
Sialic acids terminate many N- and O-glycans and are widely distributed on cell surfaces. There are a diverse range of enzymes which interact with these sugars throughout the tree of life. They can act as receptors for influenza and specific betacoronaviruses in viral binding and their cleavage is important in virion release. Sialic acids are also exploited by both commensal and pathogenic bacteria for nutrient acquisition. A common modification of sialic acid is 9-O-acetylation, which can limit the action of sialidases. Some bacteria, including human endosymbionts, employ esterases to overcome this modification. However, few bacterial sialic acid 9-O-acetylesterases (9-O-SAEs) have been structurally characterized. Here, the crystal structure of a 9-O-SAE from Phocaeicola vulgatus (PvSAE) is reported. The structure of PvSAE was determined to resolutions of 1.44 and 2.06 Å using crystals from two different crystallization conditions. Structural characterization revealed PvSAE to be a dimer with an SGNH fold, named after the conserved sequence motif of this family, and a Ser–His–Asp catalytic triad. These structures also reveal flexibility in the most N-terminal α-helix, which provides a barrier to active-site accessibility. Biochemical assays also show that PvSAE deacetylates both mucin and the acetylated chromophore para-nitrophenyl acetate. This structural and biochemical characterization of PvSAE furthers the understanding of 9-O-SAEs and may aid in the discovery of small molecules targeting this class of enzyme.
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Scott, Hannah, Gideon J. Davies, and Zachary Armstrong. "The structure of Phocaeicola vulgatus sialic acid acetylesterase." Acta Crystallographica Section D Structural Biology 78, no. 5 (April 26, 2022): 647–57. http://dx.doi.org/10.1107/s2059798322003357.
Full textAbstract:
Sialic acids terminate many N- and O-glycans and are widely distributed on cell surfaces. There are a diverse range of enzymes which interact with these sugars throughout the tree of life. They can act as receptors for influenza and specific betacoronaviruses in viral binding and their cleavage is important in virion release. Sialic acids are also exploited by both commensal and pathogenic bacteria for nutrient acquisition. A common modification of sialic acid is 9-O-acetylation, which can limit the action of sialidases. Some bacteria, including human endosymbionts, employ esterases to overcome this modification. However, few bacterial sialic acid 9-O-acetylesterases (9-O-SAEs) have been structurally characterized. Here, the crystal structure of a 9-O-SAE from Phocaeicola vulgatus (PvSAE) is reported. The structure of PvSAE was determined to resolutions of 1.44 and 2.06 Å using crystals from two different crystallization conditions. Structural characterization revealed PvSAE to be a dimer with an SGNH fold, named after the conserved sequence motif of this family, and a Ser–His–Asp catalytic triad. These structures also reveal flexibility in the most N-terminal α-helix, which provides a barrier to active-site accessibility. Biochemical assays also show that PvSAE deacetylates both mucin and the acetylated chromophore para-nitrophenyl acetate. This structural and biochemical characterization of PvSAE furthers the understanding of 9-O-SAEs and may aid in the discovery of small molecules targeting this class of enzyme.
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Mahapatra, Sebabrata, Tetsuya Yagi, John T. Belisle, Benjamin J. Espinosa, Preston J. Hill, Michael R. McNeil, Patrick J. Brennan, and Dean C. Crick. "Mycobacterial Lipid II Is Composed of a Complex Mixture of Modified Muramyl and Peptide Moieties Linked to Decaprenyl Phosphate." Journal of Bacteriology 187, no. 8 (April 15, 2005): 2747–57. http://dx.doi.org/10.1128/jb.187.8.2747-2757.2005.
Full textAbstract:
ABSTRACT Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-l-alanine-d-glutamate-meso-diaminopimelic acid-d-alanyl-d-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal d-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.
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Bennick, A. "Structural and Genetic Aspects of Proline-rich Proteins." Journal of Dental Research 66, no. 2 (February 1987): 457–61. http://dx.doi.org/10.1177/00220345870660021201.
Full textAbstract:
Considerable advances have been made in the genetics of salivary proline-rich proteins (PRP). The genes for acidic, basic, and glycosylated PRP have been cloned. They code for precursor proteins that all have an acidic N-terminal followed by proline-rich repeat sequences. Structural studies on secreted proteins have demonstrated that not only acidic but also some basic PRPs have this general structure. It is possible that mRNA for different PRP may have originated from a single gene by differential mRNA splicing, but post-translational cleavages of the primary translation product apparently also occur. In vitro translation of salivary gland mRNA results in a single precursor protein for acidic PRP. Such in vitro translated protein can be cleaved by salivary kallikrein, giving rise to two commonly secreted acidic PRPs, and kallikrein or kallikrein-like enzymes may be responsible for other post-translational cleavages of PRPs. Acidic as well as some basic PRPs are phosphorylated. A protein kinase has been demonstrated in salivary glands which phosphorylates the PRPs and other secreted salivary proteins in a cAMP and Ca2+-calmodulinindependent manner. Knowledge of the conformation of PRPs is limited. There is no conclusive evidence of polyproline-like structure in the proline-rich part of PRPs. Ca2+ binding studies on acidic PRPs indicate that there is interaction between the Ca2+ binding N-terminal end and the proline-rich C-terminal part. This interaction is relieved by modification of arginine side-chains. 1H, 32P, and 43Ca NMR studies have further elucidated the conformation of acidic PRPs in solution. Present evidence shows that salivary PRPs constitute a unique superfamily of proteins which pose a number of interesting questions concerning gene structure, pre- and post-translational modifications, and protein conformation.
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RANGARAJAN, Minnie, Susan J. M. SMITH, Sally U, and Michael A. CURTIS. "Biochemical characterization of the arginine-specific proteases of Porphyromonas gingivalis W50 suggests a common precursor." Biochemical Journal 323, no. 3 (May 1, 1997): 701–9. http://dx.doi.org/10.1042/bj3230701.
Full textAbstract:
Extracellular proteases of Porphyromonas gingivalis specific for arginyl peptide bonds are considered to be important virulence factors in periodontal disease. In order to determine the number, inter-relationship and kinetic properties of these proteases, extracellular enzymes with this peptide-bond specificity were purified and characterized from P. gingivalis W50. Three forms, which we denote RI, RI-A and RI-B, accounted for all of the activity in the supernatant. All three enzymes contain an α chain of ∼54 kDa with the same N-terminal amino acid sequence. RI is a heterodimer of non-covalently linked α and β chains which migrate to the same position on SDS/PAGE but which can be resolved by 8 M urea/PAGE. RI-A and RI-B are both monomeric, but the molecular mass of RI-B (70–80 kDa) is significantly increased due to post-translational modification with lipopolysaccharide. All forms show absolute specificity for peptide bonds with Arg in the P1 position and are also capable of hydrolysing N-terminal Arg and C-terminal Arg–Arg peptide bonds. Thus they show limited amino- and carboxy-peptidase activity. For the hydrolysis of Nα-benzoyl-l-Arg-p-nitroanilide, the pH optimum is 8.0 at 30 °C. The Vmax for all three enzymes is controlled by ionization of two residues with apparent pKas at 30 °C of 6.5±0.05 and 9.7±0.05, and ΔH values of ∼29 kJ/mol and ∼ 24 kJ/mol in the enzyme–substrate complex. By analogy with papain, the pKa of 6.5 could be ascribed to a Cys and the pKa of 9.7 to a His residue. E-64 [l-trans-epoxysuccinyl-leucylamide-4-(4-guanidino)butane] is a competitive inhibitor of RI, RI-A and RI-B. Based on physical properties and kinetic behaviour, RI-A appears to be analogous to gingipain from P. gingivalis HG66. However the α/β structure of RI differs significantly from that of the high-molecular-mass multimeric complex of gingipain containing four haemagglutinins described by others. Since the genes for RI and high-molecular-mass gingipain are identical, the data indicate that an alternative processing pathway is involved in the formation of RI from the initial precursor. Furthermore, the identical N-termini and enzymic properties of the catalytic component of RI, RI-A and RI-B suggest that the maturation pathway of the RI precursor may also give rise to RI-A and RI-B. The physiological functions of these isoforms and their role in the disease process may become more apparent through examination of their interactions with host proteins.
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Tulsiani, D. R. P., M. D. Skudlarek, Y. Araki, and M. C. Orgebin-Crist. "Purification and characterization of two forms of β-d-galactosidase from rat epididymal luminal fluid: evidence for their role in the modification of sperm plasma membrane glycoprotein(s)." Biochemical Journal 305, no. 1 (January 1, 1995): 41–50. http://dx.doi.org/10.1042/bj3050041.
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Previous studies from this laboratory have identified rat epididymal luminal fluid acid beta-D-galactosidase activity which also optimally hydrolyses a glycoprotein substrate at neutral pH [Skudlarek, Tulsiani and Orgebin-Crist (1992) Biochem. J. 286, 907-914]. We have now separated the luminal fluid beta-D-galactosidase into two molecular forms by ion-exchange chromatography on a column of DE-52. The separated enzyme activities were purified to an apparent homogeneity by molecular-sieve chromatography followed by affinity chromatography on a column of immobilized p-nitrophenyl beta-D-thiogalactopyranoside. The purified forms, when resolved by SDS/PAGE under reducing conditions, showed apparent molecular masses of 84 and 97 kDa. Kinetic studies, including a pH-dependent substrate preference and pH-dependent association/dissociation, disclosed no differences between these two forms. The two forms had identical N-terminal amino acid sequences. However, the 97 kDa form contained much more total carbohydrate and sialic acid than the 84 kDa form. The carbohydrate moieties in the two forms were assessed by comparing their size on SDS/PAGE before and after treatment with endo-enzymes. The removal of N-linked glycans by treatment with N-glycanase or endoglycosidase F generated de-N-glycosylated polypeptides of an apparent molecular mass of 70 kDa, and indicated that the two forms contained varying amounts of asparagine (N)-linked high mannose/hybrid-type and biantennary complex-type oligosaccharides. This result and the fact that the two molecular forms had identical N-terminal amino acid sequences indicated that the two forms probably have identical or very similar polypeptides. The potential role of the enzyme in modification of sperm plasma membrane (PM) glycoproteins was examined by resolving caput sperm PM proteins (before and after treatment in vitro of the membranes with the purified beta-D-galactosidase) on SDS/PAGE, followed by staining with peanut agglutinin (PNA), a lectin which preferentially binds to Gal beta 1,3GalNAc-linkages found in O-linked glycoproteins. The evidence presented in this report has indicated that a PNA-positive glycoprotein of an apparent molecular mass of 135-150 kDa present on the caput (but not cauda) sperm PM is degalactosylated by the digestion in vitro of the membranes with purified luminal fluid beta-D-galactosidase. This result suggests a possible role for the epididymal luminal fluid beta-D-galactosidases.
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Park, Suk-Youl, Hyun-Ju Lee, Jung-Mi Song, Jiali Sun, Hyo-Jeong Hwang, Kosuke Nishi, and Jeong-Sun Kim. "Structural characterization of a modification subunit of a putative type I restriction enzyme fromVibrio vulnificusYJ016." Acta Crystallographica Section D Biological Crystallography 68, no. 11 (October 18, 2012): 1570–77. http://dx.doi.org/10.1107/s0907444912038826.
Full textAbstract:
In multifunctional type I restriction enzymes, active methyltransferases (MTases) are constituted of methylation (HsdM) and specificity (HsdS) subunits. In this study, the crystal structure of a putative HsdM subunit fromVibrio vulnificusYJ016 (vvHsdM) was elucidated at a resolution of 1.80 Å. A cofactor-binding site forS-adenosyl-L-methionine (SAM, a methyl-group donor) is formed within the C-terminal domain of an α/β-fold, in which a number of residues are conserved, including the GxGG and (N/D)PP(F/Y) motifs, which are likely to interact with several functional moieties of the SAM methyl-group donor. Comparison with the N6 DNA MTase ofThermus aquaticusand other HsdM structures suggests that two aromatic rings (Phe199 and Phe312) in the motifs that are conserved among the HsdMs may sandwich both sides of the adenine ring of the recognition sequence so that a conserved Asn residue (Asn309) can interact with the N6 atom of the target adenine base (a methyl-group acceptor) and locate the target adenine base close to the transferred SAM methyl group.
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Burenina, O. Yu, E. A. Fedotova, A. Yu Ryazanova, A. S. Protsenko, M. V. Zakharova, A. S. Karyagina, A. S. Solonin, T. S. Oretskaya, and E. A. Kubareva. "Peculiarities of the Regulation of Gene Expression in the Ecl18kI Restriction–Modification System." Acta Naturae 5, no. 2 (June 15, 2013): 70–80. http://dx.doi.org/10.32607/20758251-2013-5-2-70-80.
Full textAbstract:
Transcription regulation in bacterial restrictionmodification (RM) systems is an important process, which provides coordinated expression levels of tandem enzymes, DNA methyltransferase (MTase) and restriction endonuclease (RE) protecting cells against penetration of alien DNA. The present study focuses on (cytosine-5)-DNA methyltransferase Ecl18kI (M.Ecl18kI), which is almost identical to DNA methyltransferase SsoII (M.SsoII) in terms of its structure and properties. Each of these enzymes inhibits expression of the intrinsic gene and activates expression of the corresponding RE gene via binding to the regulatory site in the promoter region of these genes. In the present work, complex formation of M.Ecl18kI and RNA polymerase from Escherichia сoli with the promoter regions of the MTase and RE genes is studied. The mechanism of regulation of gene expression in the Ecl18kI RM system is thoroughly investigated. M.Ecl18kI and RNA polymerase are shown to compete for binding to the promoter region. However, no direct contacts between M.Ecl18kI and RNA polymerase are detected. The properties of M.Ecl18kI and M.SsoII mutants are studied. Amino acid substitutions in the N-terminal region of M.Ecl18kI, which performs the regulatory function, are shown to influence not only M.Ecl18kI capability to interact with the regulatory site and to act as a transcription factor, but also its ability to bind and methylate the substrate DNA. The loss of methylation activity does not prevent MTase from performing its regulatory function and even increases its affinity to the regulatory site. However, the presence of the domain responsible for methylation in the M.Ecl18kI molecule is necessary for M.Ecl18kI to perform its regulatory function.
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Soupene, Eric, and Frans A. Kuypers. "Dual Role of ACBD6 in the Acylation Remodeling of Lipids and Proteins." Biomolecules 12, no. 12 (November 22, 2022): 1726. http://dx.doi.org/10.3390/biom12121726.
Full textAbstract:
The transfer of acyl chains to proteins and lipids from acyl-CoA donor molecules is achieved by the actions of diverse enzymes and proteins, including the acyl-CoA binding domain-containing protein ACBD6. N-myristoyl-transferase (NMT) enzymes catalyze the covalent attachment of a 14-carbon acyl chain from the relatively rare myristoyl-CoA to the N-terminal glycine residue of myr-proteins. The interaction of the ankyrin-repeat domain of ACBD6 with NMT produces an active enzymatic complex for the use of myristoyl-CoA protected from competitive inhibition by acyl donor competitors. The absence of the ACBD6/NMT complex in ACBD6.KO cells increased the sensitivity of the cells to competitors and significantly reduced myristoylation of proteins. Protein palmitoylation was not altered in those cells. The specific defect in myristoyl-transferase activity of the ACBD6.KO cells provided further evidence of the essential functional role of the interaction of ACBD6 with the NMT enzymes. Acyl-CoAs bound to the acyl-CoA binding domain of ACBD6 are acyl donors for the lysophospholipid acyl-transferase enzymes (LPLAT), which acylate single acyl-chain lipids, such as the bioactive molecules LPA and LPC. Whereas the formation of acyl-CoAs was not altered in ACBD6.KO cells, lipid acylation processes were significantly reduced. The defect in PC formation from LPC by the LPCAT enzymes resulted in reduced lipid droplets content. The diversity of the processes affected by ACBD6 highlight its dual function as a carrier and a regulator of acyl-CoA dependent reactions. The unique role of ACBD6 represents an essential common feature of (acyl-CoA)-dependent modification pathways controlling the lipid and protein composition of human cell membranes.
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SLEAT, David E., Stephen R. KRAUS, Istvan SOHAR, Henry LACKLAND, and Peter LOBEL. "α-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine." Biochemical Journal 324, no. 1 (May 15, 1997): 33–39. http://dx.doi.org/10.1042/bj3240033.
Full textAbstract:
Most newly synthesized lysosomal enzymes contain a transient carbohydrate modification, mannose 6-phosphate (Man-6-P), which signals their vesicular transport from the Golgi to the lysosome via Man-6-P receptors (MPRs). We have examined Man-6-P glycoproteins in human urine by using a purified soluble fragment of the soluble cation-independent MPR (sCI-MPR) as a preparative and analytical affinity reagent. In a survey of urine samples from seven healthy subjects, the pattern of Man-6-P glycoproteins detected with iodinated sCI-MPR as a probe in a blotting assay was essentially identical in each, regardless of sex or age. Two bands of approx. 100 and 110 kDa were particularly prominent. Man-6-P glycoproteins in human urine were purified by affinity chromatography on immobilized sCI-MPR. Seven distinct bands revealed by SDS/PAGE and Coomassie Blue staining were subjected to N-terminal sequence analysis. The prominent 100 and 110 kDa Man-6-P glycoproteins were identified as N-acetylglucosamine-6-sulphatase and α-glucosidase respectively. This identification was confirmed by molecular mass determinations on the two major bands after deglycosylation. Sequence analysis revealed arylsulphatase A and several previously unidentified proteins as minor species. Man-6-P glycoproteins were also purified on an analytical scale to determine the proportion of a number of lysosomal enzyme activities represented by the mannose-6-phosphorylated forms. The lysosomal enzymes in urine containing the highest proportion of mannose-6-phosphorylated form were β-mannosidase (82%), hexosaminidase (27%) and α-glucosidase (24%). The profiles of Man-6-P glycoproteins detected by blotting in urine and plasma were not similar, suggesting that the urinary species are not derived from the bloodstream.
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Oikawa, Daisuke, Kouhei Shimizu, and Fuminori Tokunaga. "Pleiotropic Roles of a KEAP1-Associated Deubiquitinase, OTUD1." Antioxidants 12, no. 2 (February 1, 2023): 350. http://dx.doi.org/10.3390/antiox12020350.
Full textAbstract:
Protein ubiquitination, which is catalyzed by ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin ligases, is a crucial post-translational modification to regulate numerous cellular functions in a spatio–temporal-specific manner. The human genome encodes ~100 deubiquitinating enzymes (DUBs), which antagonistically regulate the ubiquitin system. OTUD1, an ovarian tumor protease (OTU) family DUB, has an N-terminal-disordered alanine-, proline-, glycine-rich region (APGR), a catalytic OTU domain, and a ubiquitin-interacting motif (UIM). OTUD1 preferentially hydrolyzes lysine-63-linked ubiquitin chains in vitro; however, recent studies indicate that OTUD1 cleaves various ubiquitin linkages, and is involved in the regulation of multiple cellular functions. Thus, OTUD1 predominantly functions as a tumor suppressor by targeting p53, SMAD7, PTEN, AKT, IREB2, YAP, MCL1, and AIF. Furthermore, OTUD1 regulates antiviral signaling, innate and acquired immune responses, and cell death pathways. Similar to Nrf2, OTUD1 contains a KEAP1-binding ETGE motif in its APGR and regulates the reactive oxygen species (ROS)-mediated oxidative stress response and cell death. Importantly, in addition to its association with various cancers, including multiple myeloma, OTUD1 is involved in acute graft-versus-host disease and autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and ulcerative colitis. Thus, OTUD1 is an important DUB as a therapeutic target for a variety of diseases.