TY - JOUR
T1 - Exploring sequence requirements for C3/C4 carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase
T2 - Insights into plasticity of the AmpC β-lactamase
AU - Drawz, Sarah M.
AU - Taracila, Magdalena
AU - Caselli, Emilia
AU - Prati, Fabio
AU - Bonomo, Robert A.
PY - 2011/6
Y1 - 2011/6
N2 - 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 C3/C4 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/C4 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/C4 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 C3/C4 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 C3/C4 carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.
AB - 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 C3/C4 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/C4 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/C4 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 C3/C4 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 C3/C4 carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.
KW - AmpC cephalosporinases
KW - Pseudomonas aeruginosa
KW - β-lactamase inhibitor resistance
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U2 - 10.1002/pro.612
DO - 10.1002/pro.612
M3 - Article
C2 - 21404358
AN - SCOPUS:79956206767
SN - 0961-8368
VL - 20
SP - 941
EP - 958
JO - Protein Science
JF - Protein Science
IS - 6
ER -