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High adaptability of the omega loop underlies the substrate-spectrum-extension evolution of a class A ?-lactamase, PenL.

ABSTRACT: The omega loop in ?-lactamases plays a pivotal role in substrate recognition and catalysis, and some mutations in this loop affect the adaptability of the enzymes to new antibiotics. Various mutations, including substitutions, deletions, and intragenic duplications resulting in tandem repeats (TRs), have been associated with ?-lactamase substrate spectrum extension. TRs are unique among the mutations as they cause severe structural perturbations in the enzymes. We explored the process by which TRs are accommodated in order to test the adaptability of the omega loop. Structures of the mutant enzymes showed that the extra amino acid residues in the omega loop were freed outward from the enzyme, thereby maintaining the overall enzyme integrity. This structural adjustment was accompanied by disruptions of the internal ?-helix and hydrogen bonds that originally maintained the conformation of the omega loop and the active site. Consequently, the mutant enzymes had a relaxed binding cavity, allowing for access of new substrates, which regrouped upon substrate binding in an induced-fit manner for subsequent hydrolytic reactions. Together, the data demonstrate that the design of the binding cavity, including the omega loop with its enormous adaptive capacity, is the foundation of the continuous evolution of ?-lactamases against new drugs.

SUBMITTER: Yi H

PROVIDER: S-EPMC5101513 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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High adaptability of the omega loop underlies the substrate-spectrum-extension evolution of a class A β-lactamase, PenL.

Yi Hyojeong H Choi Jin Myung JM Hwang Junghyun J Prati Fabio F Cao Thinh-Phat TP Lee Sung Haeng SH Kim Heenam Stanley HS

Scientific reports 20161109

The omega loop in β-lactamases plays a pivotal role in substrate recognition and catalysis, and some mutations in this loop affect the adaptability of the enzymes to new antibiotics. Various mutations, including substitutions, deletions, and intragenic duplications resulting in tandem repeats (TRs), have been associated with β-lactamase substrate spectrum extension. TRs are unique among the mutations as they cause severe structural perturbations in the enzymes. We explored the process by which T ...[more]

PMID: 27827433

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Project description:KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25-1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

Dataset Information

High adaptability of the omega loop underlies the substrate-spectrum-extension evolution of a class A ?-lactamase, PenL.

Publications

High adaptability of the omega loop underlies the substrate-spectrum-extension evolution of a class A β-lactamase, PenL.

Similar Datasets

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