Role of the Conserved Disulfide Bridge in Class A Carbapenemases

Clyde A Smith,Zahra Nossoni,Marta Toth,Nichole K Stewart,Hilary Frase,Sergei B Vakulenko

doi:10.1074/jbc.m116.749648

Abstract

Some members of the class A β-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 Cys69 and Cys238 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 β-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 hydrogen-bonding 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 β-lactamases. This would likely apply to all other class A carbapenemases given the high degree of their structural similarity.

Highlights

␤-Lactamases are enzymes produced by bacteria for self-protection against various ␤-lactam antibiotics [1]
To counter the growing problem of resistance, novel ␤-lactams such as expanded spectrum cephalosporins and ␤-lactamase inhibitors were developed. Extensive use of these drugs for treatment of infectious diseases triggered the selection in Gram-negative bacteria of variants of class A TEMand SHV-type ␤-lactamases capable of protecting the microorganisms against these newer antimicrobial agents [5]
Based on a comparison of the homology models of the NMCA and SME-1 carbapenemases with the structure of the class A ␤-lactamase, TEM-1, that is devoid of carbapenemase activity, it was proposed that presence of the disulfide bridge changes the geometry of the active site resulting in carbapenemase activity [15]

Summary

Introduction

␤-Lactamases are enzymes produced by bacteria for self-protection against various ␤-lactam antibiotics [1]. To counter the growing problem of resistance, novel ␤-lactams such as expanded spectrum cephalosporins (including cefotaxime, ceftriaxone, and ceftazidime) and ␤-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) were developed Extensive use of these drugs for treatment of infectious diseases triggered the selection in Gram-negative bacteria of variants of class A TEMand SHV-type ␤-lactamases capable of protecting the microorganisms against these newer antimicrobial agents [5]. We substituted conserved cysteine residues in the class A ␤-lactamases GES-1, GES-2, GES-5, NMCA, SFC-1, and SME-1 and evaluated how the resulting disruption of the disulfide bridge affects activity of these enzymes against the carbapenem imipenem and other ␤-lactam antibiotics These experiments, combined with structural studies, allowed us to elucidate the role of the disulfide bridge in the class A carbapenemases

Methods

Results

Conclusion