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Different intermediate populations formed by tazobactam, sulbactam, and clavulanate reacting with SHV-1 beta-lactamases: Raman crystallographic evidence.

ABSTRACT: Tazobactam, sulbactam, and clavulanic acid are the only beta-lactamase inhibitors in clinical use. Comparative inhibitory activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases conclude that tazobactam is superior to both clavulanic acid and sulbactam. Thus far, the majority of explanations for this phenomenon have relied on kinetic studies, which report differences in the ligands' apparent dissociation constants and number of turnovers before inactivation. Due their innate limitations, these investigations do not examine the identity of intermediates on the reaction pathway and relate them to the efficacy of the inhibitors. In the present study, the reactions between the three inhibitors and SHV-1 beta-lactamase have been examined in single crystals using a Raman microscope. The results show that tazobactam forms a predominant population of trans-enamine, a chemically inert species, with SHV-1, while clavulanate and sulbactam form a mixture of trans-enamine and two labile species, the cis-enamine and imine. The same reactions are then reexamined using a deacylation-deficient variant, SHV E166A, that has been used to trap acyl-enzyme intermediates for X-ray crystallographic analysis. Our Raman data show that significant differences exist between the wild-type and SHV E166A acyl-enzyme populations. Namely, compared to SHV-1, sulbactam shows significantly smaller populations of cis-enamine and imine in the E166A variant, while clavulanate exists almost exclusively as trans-enamine in the E166A active site. Using clavulanate as an example, we also show that Raman crystallography can provide novel information on the presence of multiple conformers or tautomers for intermediates within a complex reaction pathway. These insights caution against the interpretation of experimental data obtained with deacylation-deficient beta-lactamases to make mechanistic conclusions about inhibitors within the enzyme.

SUBMITTER: Kalp M

PROVIDER: S-EPMC2661619 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Different intermediate populations formed by tazobactam, sulbactam, and clavulanate reacting with SHV-1 beta-lactamases: Raman crystallographic evidence.

Kalp Matthew M Totir Monica A MA Buynak John D JD Carey Paul R PR

Journal of the American Chemical Society 20090201 6

Tazobactam, sulbactam, and clavulanic acid are the only beta-lactamase inhibitors in clinical use. Comparative inhibitory activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases conclude that tazobactam is superior to both clavulanic acid and sulbactam. Thus far, the majority of explanations for this phenomenon have relied on kinetic studies, which report differences in the ligands' apparent dissociation constants and number of turnovers before inact ...[more]

PMID: 19161282

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Project description:β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M(-1) s(-1), versus 60,300 M(-1) s(-1) for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15-avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A β-lactamases, S130 may be the most important amino acid for the inhibition of class A β-lactamases, perhaps even for the novel diazabicyclooctane class of β-lactamase inhibitors.

Project description:BACKGROUND: SHV ?-lactamases confer resistance to a broad range of antibiotics by accumulating mutations. The number of SHV variants is steadily increasing. 117 SHV variants have been assigned in the SHV mutation table (http://www.lahey.org/Studies/). Besides, information about SHV ?-lactamases can be found in the rapidly growing NCBI protein database. The SHV ?-Lactamase Engineering Database (SHVED) has been developed to collect the SHV ?-lactamase sequences from the NCBI protein database and the SHV mutation table. It serves as a tool for the detection and reconciliation of inconsistencies, and for the identification of new SHV variants and amino acid substitutions. DESCRIPTION: The SHVED contains 200 protein entries with distinct sequences and 20 crystal structures. 83 protein sequences are included in the both the SHV mutation table and the NCBI protein database, while 35 and 82 protein sequences are only in the SHV mutation table and the NCBI protein database, respectively. Of these 82 sequences, 41 originate from microbial sources, and 22 of them are full-length sequences that harbour a mutation profile which has not been classified yet in the SHV mutation table. 27 protein entries from the NCBI protein database were found to have an inconsistency in SHV name identification. These inconsistencies were reconciled using information from the SHV mutation table and stored in the SHVED.The SHVED is accessible at http://www.LacED.uni-stuttgart.de/classA/SHVED/. It provides sequences, structures, and a multisequence alignment of SHV ?-lactamases with the corrected annotation. Amino acid substitutions at each position are also provided. The SHVED is updated monthly and supplies all data for download. CONCLUSIONS: The SHV ?-Lactamase Engineering Database (SHVED) contains information about SHV variants with reconciled annotation. It serves as a tool for detection of inconsistencies in the NCBI protein database, helps to identify new mutations resulting in new SHV variants, and thus supports the investigation of sequence-function relationships of SHV ?-lactamases.

Project description:Sulbactam is a mechanism-based inhibitor of beta-lactamase enzymes used in clinical practice. It undergoes a complex series of chemical reactions in the active site that have been studied extensively in the past three decades. However, the actual species that gives rise to inhibition in a clinical setting has not been established. Recent studies by our group, using Raman microscopy and X-ray crystallography, have found that large quantities of enamine-based acyl-enzyme species are present within minutes in single crystals of SHV-1 beta-lactamases which can lead to significant inhibition. The enamines are formed by breakdown of the cyclic beta-lactam structures with further transformations leading to imine formation and subsequent isomerization to cis and/or trans enamines. Another favored form of inhibition arises from attack on the imine by a second nucleophilic amino acid side chain, e.g., from serine 130, to form a cross-linked species in the active site that can degrade to an acrylate-like species irreversibly bound to the enzyme. Thus, the imine is at a branch point on the reaction pathway. Using sulbactam and 6,6-dideuterated sulbactam we follow these alternate paths in WT and E166A SHV-1 beta-lactamase by means of Raman microscopic studies on single enzyme crystals. For the unlabeled sulbactam, the Raman data show the presence of an acrylate-like species, probably 3-serine acrylate, several hours after the reaction is started in the crystal. However, for the 6,6-dideutero analogue the acrylate signature appears on the time scale of minutes. The Raman signatures, principally an intense feature near 1530 cm-1, are assigned based on quantum mechanical calculations on model compounds that mimic acrylate species in the active site. The different time scales observed for acrylate-like product formation are ascribed to different rates of reaction involving the imine intermediate. It is proposed that for the unsubstituted sulbactam the conversion from imine to enamine, which involves breaking a C-H bond, is aided by quantum mechanical tunneling. For the 6,6-dideutero-sulbactam the same step involves breaking a C-D bond, which has little or no assistance from tunneling. Consequently the conversion to enamines is slower, and a higher population of imine results, presenting the opportunity for the competing reaction with the second nucleophile, serine 130 being the prime candidate. The hydrolysis of the resulting cross-linked intermediate leads to the observed rapid buildup of the acrylate product in the Raman spectra from the dideutero analogue. The protocol used here, essentially running the reactions with the two forms of sulbactam in parallel, provides an element of control and enables us to conclude that, for the unsubstituted sulbactam, the formation of the cross-linked intermediate and the final irreversible acrylate product is not a significant route to inhibition of SHV-1.

Dataset Information

Different intermediate populations formed by tazobactam, sulbactam, and clavulanate reacting with SHV-1 beta-lactamases: Raman crystallographic evidence.

Publications

Different intermediate populations formed by tazobactam, sulbactam, and clavulanate reacting with SHV-1 beta-lactamases: Raman crystallographic evidence.

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