Premium
Exploring sequence requirements for C 3 /C 4 carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase: Insights into plasticity of the AmpC β‐lactamase
Author(s) -
Drawz Sarah M.,
Taracila Magdalena,
Caselli Emilia,
Prati Fabio,
Bonomo Robert A.
Publication year - 2011
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.612
Subject(s) - carboxylate , pseudomonas aeruginosa , penicillin binding proteins , lactam , active site , enzyme , biology , stereochemistry , cephalosporin , cephalosporin c , biochemistry , penicillin , chemistry , antibiotics , genetics , bacteria
Abstract In Pseudomonas aeruginosa , the chromosomally encoded class C cephalosporinase (AmpC β‐lactamase) is often responsible for high‐level resistance to β‐lactam antibiotics. Despite years of study of these important β‐lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas ‐derived cephalosporinase (PDC) remains scarce. Insights into structure‐function relationships are crucial to the design of both β‐lactams and high‐affinity inhibitors. In order to understand how PDC recognizes the C 3 /C 4 carboxylate of β‐lactams, we first examined a molecular model of a P. aeruginosa AmpC β‐lactamase, PDC‐3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C 3 /C 4 carboxylate characteristic of β‐lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C 3 /C 4 β‐lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC‐3 β‐lactamase maintains a high level of activity despite the substitution of C 3 /C 4 β‐lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC‐3 enzyme and the Arg349Ala variant. Our examination of the PDC‐3 active site revealed that more than one residue could serve to interact with the C 3 /C 4 carboxylate of the β‐lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.