Abstract
The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition. Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme. After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.
Highlights
From the ‡Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, §Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, and the ¶Department of Biochemistry, Case School of Medicine, Cleveland, Ohio 44106
The mass increases observed for SHV-1 and Ser130 3 Gly (؉ ⌬ 88 Da and ؉ ⌬ 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive -lactamase
The molecular mass of the apoenzyme and tazobactam-inhibited SHV-1 -lactamases measured by Electrospray ionization mass spectrometry (ESI/MS) are 28,872 Ϯ 3 and 28,959 Ϯ 3 Da, respectively
Summary
The mass increases observed for SHV-1 and Ser130 Gly (؉ ⌬ 88 Da and ؉ ⌬ 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive -lactamase. These two mass increments are consistent with the formation of an aldehyde (؉ ⌬ 70 Da) and a hydrated aldehyde (؉ ⌬ 88 Da) as stable products of inhibition. By examining the inhibitor-resistant Ser130 Gly -lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser in this enzyme. Lowercase letters a– d refer to theoretical peptides from trypsin digestion that were not identified in these experiments
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