The Basis for Carbapenem Hydrolysis by Class A β-Lactamases: A Combined Investigation using Crystallography and Simulations

Fonseca, Fátima; Chudyk, Ewa I.; Kamp, Marc W. van der; Correia, António; Mulholland, Adrian J.; Spencer, James

doi:10.1021/ja304460j

Cited by 84 publications

(157 citation statements)

References 76 publications

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“…The poorer turnover for the carbapenems results from interference with the approach of the hydrolytic water to the acyl-enzyme by the 6␣-hydroxyethyl group of the carbapenem (56)(57)(58). Binding of carbapenems also causes conformational changes within the active site of the ␤-lactamase and particularly affects the residues in the Ser-X-Asn motif (e.g., Ser130 and Asn132 in TEM and SHV enzymes) and the residues of loop 1 (e.g., Tyr105).…”

Section: Discussionmentioning

confidence: 99%

Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

Smith

Kumarasiri

Zhang

et al. 2013

Antimicrob Agents Chemother

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b Penicillin-binding protein 5 (PBP5) is one of the most abundant PBPs in Pseudomonas aeruginosa. Although its main function is that of a cell wall DD-carboxypeptidase, it possesses sufficient ␤-lactamase activity to contribute to the ability of P. aeruginosa to resist the antibiotic activity of the ␤-lactams. The study of these dual activities is important for understanding the mechanisms of antibiotic resistance by P. aeruginosa, an important human pathogen, and to the understanding of the evolution of ␤-lactamase activity from the PBP enzymes. We purified a soluble version of P. aeruginosa PBP5 (designated Pa sPBP5) by deletion of its C-terminal membrane anchor. Under in vitro conditions, Pa sPBP5 demonstrates both DD-carboxypeptidase and expanded-spectrum ␤-lactamase activities. Its crystal structure at a 2.05-Å resolution shows features closely resembling those of the class A ␤-lactamases, including a shortened loop spanning residues 74 to 78 near the active site and with respect to the conformations adopted by two active-site residues, Ser101 and Lys203. These features are absent in the related PBP5 of Escherichia coli. A comparison of the two Pa sPBP5 monomers in the asymmetric unit, together with molecular dynamics simulations, revealed an active-site flexibility that may explain its carbapenemase activity, a function that is absent in the E. coli PBP5 enzyme. Our functional and structural characterizations underscore the versatility of this PBP5 in contributing to the ␤-lactam resistance of P. aeruginosa while highlighting how broader ␤-lactamase activity may be encoded in the structural folds shared by the PBP and serine ␤-lactamase classes.

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Section: Discussionmentioning

confidence: 99%

Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

Smith

Kumarasiri

Zhang

et al. 2013

Antimicrob Agents Chemother

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“…The serine-to-alanine substitution at position 237 decreased the k cat values for imipenem; cephalothin hydrolysis rates were reduced by a factor of 5, whereas benzylpenicillin k cat values remained unchanged (150). However, no single residue has yet been identified as being responsible for carbapenem resistance (147,151). With the exception of KPC types, all types (BIC, FRI, IMI, NMC, SFC, and SME) have an additional residue at position 139a (Glu) (Fig.…”

Section: Toca P Vulgaris R Aquatilis and S Fonticola)mentioning

confidence: 99%

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

Slama²,

et al. 2016

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SUMMARYFor medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. This is particularly true for data concerning class A β-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. Many genomes have been reported to contain putative β-lactamase genes, which can be compared with representative types. We analyzed several hundred amino acid sequences of class A β-lactamase enzymes for phylogenic relationships, the presence of specific residues, and cluster patterns. A clear distinction was first made between dd-peptidases and class A enzymes based on a small number of residues (S70, K73, P107, 130SDN132, G144, E166, 234K/R, 235T/S, and 236G [Ambler numbering]). Other residues clearly separated two main branches, which we named subclasses A1 and A2. Various clusters were identified on the major branch (subclass A1) on the basis of signature residues associated with catalytic properties (e.g., limited-spectrum β-lactamases, extended-spectrum β-lactamases, and carbapenemases). For subclass A2 enzymes (e.g., CfxA, CIA-1, CME-1, PER-1, and VEB-1), 43 conserved residues were characterized, and several significant insertions were detected. This diversity in the amino acid sequences of β-lactamases must be taken into account to ensure that new enzymes are accurately identified. However, with the exception of PER types, this diversity is poorly represented in existing X-ray crystallographic data.

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“…Comparison of the same two domains (domain 1, residues 24 -65 and 217-291; domain 2, residues 66 -216; standard Ambler numbering (14)) of the wild-type structures of SME-1 (PDB code 1DY6) (24), NMCA (PDB code 1BUE) (25), and SFC-1 (PDB code 4EQI) (10) gives approximately the same buried surface area at the interface (2850 -2900 Å 2 ). Importantly, however, there are less hydrogen bonding interactions between the two structural domains in SME-1, NMCA, and SFC-1, both protein-protein and water-mediated, and the subsequent decrease in interdomain hydrogen bonding energy may be sufficient to destabilize the interface following the loss of the disulfide bond.…”

Section: Ges-5 C69g Structure and Functionmentioning

confidence: 99%

Role of the Conserved Disulfide Bridge in Class A Carbapenemases

Smith

Nossoni

Tóth

et al. 2016

Journal of Biological Chemistry

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Some members of the class A ␤-lactamase family are capable of conferring resistance to the last resort antibiotics, carbapenems. A unique structural feature of these clinically important enzymes, collectively referred to as class A carbapenemases, is a disulfide bridge between invariant Cys 69 and Cys 238 residues. It was proposed that this conserved disulfide bridge is responsible for their carbapenemase activity, but this has not yet been validated. Here we show that disruption of the disulfide bridge in the GES-5 carbapenemase by the C69G substitution results in only minor decreases in the conferred levels of resistance to the carbapenem imipenem and other ␤-lactams. Kinetic and circular dichroism experiments with C69G-GES-5 demonstrate that this small drop in antibiotic resistance is due to a decline in the enzyme activity caused by a marginal loss of its thermal stability. The atomic resolution crystal structure of C69G-GES-5 shows that two domains of this disulfide bridge-deficient enzyme are held together by an intensive hydrogen-bonding network. As a result, the protein architecture and imipenem binding mode remain unchanged. In contrast, the corresponding hydrogenbonding networks in NMCA, SFC-1, and SME-1 carbapenemases are less intensive, and as a consequence, disruption of the disulfide bridge in these enzymes destabilizes them, which causes arrest of bacterial growth. Our results demonstrate that the disulfide bridge is essential for stability but does not play a direct role in the carbapenemase activity of the GES family of ␤-lactamases. This would likely apply to all other class A carbapenemases given the high degree of their structural similarity.

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The Basis for Carbapenem Hydrolysis by Class A β-Lactamases: A Combined Investigation using Crystallography and Simulations

Cited by 84 publications

References 76 publications

Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

Role of the Conserved Disulfide Bridge in Class A Carbapenemases

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