Neisseria gonorrhoeae Penicillin-Binding Protein 3 Exhibits Exceptionally High Carboxypeptidase and β-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.

Dual Roles of FmtA in Staphylococcus aureus Cell Wall Biosynthesis and Autolysis

“…In comparison, the catalytic efficiency of the DD-carboxypeptidase activity of PBP4 of S. aureus was determined to be 15.7 M Ϫ1 s Ϫ1 (30). The activity of other well-studied PBPs has also been reported to be higher, ranging from 10 M Ϫ1 s Ϫ1 for PBP5 of E. coli to 2.9 ϫ 10 4 M Ϫ1 s Ϫ1 for PBP3 of N. gonorrhoeae, according to a study using the same substrate as used in our study (42).…”

“…It is part of the core cell wall stimulon (26). Its expression level is elevated by the presence of cell wall inhibitors and knockout of genes that are directly involved with peptidoglycan biosynthesis (3,23,26,27,42,46). Furthermore, inactivation of fmtA results in a lack of WTAs on S. aureus cell walls and deficiency in biofilm production (4,44).…”

Identification of Amino Acids Conferring High-Level Resistance to Expanded-Spectrum Cephalosporins in the penA Gene from Neisseria gonorrhoeae Strain H041

“…k 2 /K s constants, which are a direct measure of the ability of an antibiotic to inhibit a PBP (32), were calculated from first-order rates of acylation of purified, soluble PBP2 variants by [ 14 C]penicillin G (Moravek, Brea, CA), as previously described (25,31,33 …”

Substrate Specificity of Low-Molecular Mass Bacterial dd-Peptidases

“…The purified enzymes were prepared as described: the Actinomadura R39 DD-peptidase, 28 Escherichia coli PBP5 (EcPBP5), 29 N. gonorrhoeae PBP 3 (NgPBP3) 30 and PBP4 (NgPBP4), 31 Bacillus subtilis PBP4a (BsPBP4a), 32 and S. pneumoniae PBP3 (SpPBP3). 33 The following peptides were purchased commercially and used as received: 10 (NeoMPS), 3 and 13 (Sigma-Aldrich), and 14−19 (New England Peptide).…”