Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4.
The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the beta-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for beta-lactam binding, DD-carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30,000 m-1.s-1). Against a set of five alpha- and epsilon-substituted l-Lys-D-Ala-D-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N epsilon-acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N epsilon-Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site-directed agents, N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac2-l-Lys-D-Ala-d-Ala (kcat/Km) was bell-shaped, with pKa values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other DD-carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs.
Stefanova
Miglena E
ME
Division of Pharmaceutical Sciences, University of Missouri-Kansas City 64110, USA.
Tomberg
Joshua
J
Davies
Christopher
C
Nicholas
Robert A
RA
Gutheil
William G
WG
D000215:Acylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017124:Conserved Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007769:Lactams / Q000378:metabolism; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009344:Neisseria gonorrhoeae / Q000201:enzymology*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007769:Lactams; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase