title: Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities.
abstract: A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.
authors.affiliation: Division of Pharmaceutical Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
authors.forename: Miglena E
authors.initials: ME
authors.lastname: Stefanova
authors.forename: Joshua
authors.initials: J
authors.lastname: Tomberg
authors.forename: Melanie
authors.initials: M
authors.lastname: Olesky
authors.forename: Joachim-Volker
authors.initials: JV
authors.lastname: Höltje
authors.forename: William G
authors.initials: WG
authors.lastname: Gutheil
authors.forename: Robert A
authors.initials: RA
authors.lastname: Nicholas
mesh_terms: D000215:Acylation; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006602:Hexosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009344:Neisseria gonorrhoeae / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D047090:beta-Lactams / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C478704:FtsI protein, E coli; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046928:Peptidoglycan Glycosyltransferase; D010450:Endopeptidases; D002267:Muramoylpentapeptide Carboxypeptidase