PMID:14661974

Citation	Stefanova, ME, Tomberg, J, Olesky, M, Höltje, JV, Gutheil, WG and Nicholas, RA (2003) Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities. Biochemistry 42:14614-25
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.
Links	PubMed Online version:10.1021/bi0350607
Keywords	Acylation; Anti-Bacterial Agents/chemistry; Anti-Bacterial Agents/metabolism; Bacterial Proteins/antagonists & inhibitors; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Carrier Proteins/antagonists & inhibitors; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; Cell Division/genetics; Cell Survival/genetics; Cloning, Molecular; Drug Resistance, Microbial; Endopeptidases/chemistry; Endopeptidases/metabolism; Enzyme Stability; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Hexosyltransferases/antagonists & inhibitors; Hexosyltransferases/chemistry; Hexosyltransferases/genetics; Hexosyltransferases/metabolism; Hydrogen-Ion Concentration; Microscopy, Electron, Scanning; Molecular Sequence Data; Muramoylpentapeptide Carboxypeptidase/antagonists & inhibitors; Muramoylpentapeptide Carboxypeptidase/chemistry; Muramoylpentapeptide Carboxypeptidase/genetics; Muramoylpentapeptide Carboxypeptidase/metabolism; Neisseria gonorrhoeae/enzymology; Neisseria gonorrhoeae/growth & development; Neisseria gonorrhoeae/metabolism; Neisseria gonorrhoeae/ultrastructure; Penicillin-Binding Proteins; Peptidoglycan Glycosyltransferase; Peptidyl Transferases/antagonists & inhibitors; Peptidyl Transferases/chemistry; Peptidyl Transferases/genetics; Peptidyl Transferases/metabolism; Protein Binding; Substrate Specificity; beta-Lactams/chemistry; beta-Lactams/metabolism
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