Title: Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases

Abstract

Carbapenems are the last resort antibiotics for treatment of life-threatening infections. The GES β-lactamases are important contributors to carbapenem resistance in clinical bacterial pathogens. A single amino acid difference at position 170 of the GES-1, GES-2, and GES-5 enzymes is responsible for the expansion of their substrate profile to include carbapenem antibiotics. This highlights the increasing need to understand the mechanisms by which the GES β-lactamases function to aid in development of novel therapeutics. We demonstrate that the catalytic efficiency of the enzymes with carbapenems meropenem, ertapenem, and doripenem progressively increases (100-fold) from GES-1 to -5, mainly due to an increase in the rate of acylation. The data reveal that while acylation is rate limiting for GES-1 and GES-2 for all three carbapenems, acylation and deacylation are indistinguishable for GES-5. The ertapenem–GES-2 crystal structure shows that only the core structure of the antibiotic interacts with the active site of the GES-2 β-lactamase. The identical core structures of ertapenem, doripenem, and meropenem are likely responsible for the observed similarities in the kinetics with these carbapenems. The lack of a methyl group in the core structure of imipenem may provide a structural rationale for the increase in turnover of this carbapenem bymore » the GES β-lactamases. As a result, our data also show that in GES-2 an extensive hydrogen-bonding network between the acyl-enzyme complex and the active site water attenuates activation of this water molecule, which results in poor deacylation by this enzyme.« less

Authors:

Stewart, Nichole K. ^[1]; Smith, Clyde A. ^[2]; Frase, Hilary ^[1]; Black, D. J. ^[3]; Vakulenko, Sergei B. ^[1]

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+ Show Author Affiliations

1. Univ. of Notre Dame, Notre Dame, IN (United States)
2. Stanford Univ., Menlo Park, CA (United States)
3. Bio-Logic USA, Knoxville, TN (United States)

Publication Date:

Mon Dec 08 00:00:00 EST 2014

Research Org.:

University of Notre Dame, IN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Inst. of Health (NIH) (United States)

OSTI Identifier:

1347218

Grant/Contract Number:  

AI089726; P41 RR001209

Resource Type:

Accepted Manuscript

Journal Name:

Biochemistry

Additional Journal Information:

Journal Volume: 54; Journal Issue: 2; Journal ID: ISSN 0006-2960

Publisher:

American Chemical Society (ACS)

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES