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The importance of the trans-enamine intermediate as a ?-lactamase inhibition strategy probed in inhibitor-resistant SHV ?-lactamase variants.

ABSTRACT: The ability of bacteria to express inhibitor-resistant (IR) ?-lactamases is stimulating the development of novel inhibitors of these enzymes. The 2'?-glutaroxypenicillinate sulfone, SA2-13, was previously designed to enhance the stabilization of the deacylation-refractory, trans-enamine inhibitory intermediate. To test whether this mode of inhibition can overcome different IR mutations, we determined the binding mode of SA2-13 through X-ray crystallography, obtaining co-crystals of the inhibitor-protein complex by soaking crystals of the IR sulfhydryl variable (SHV) ?-lactamase variants S130G and M69V with the inhibitor. The 1.45 Å crystal structure of the S130G?SHV:SA2-13 complex reveals that SA2-13 is still able to form the stable trans-enamine intermediate similar to the wild-type complex structure, yet with its carboxyl linker shifted deeper into the active site in the space vacated by the S130G mutation. In contrast, data from crystals of the M69V SHV:SA2-13 complex at 1.3 Å did not reveal clear inhibitor density indicating that this IR variant disfavors the trans-enamine conformation, likely due to a subtle shift in A237.

SUBMITTER: Ke W

PROVIDER: S-EPMC3523351 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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The importance of the trans-enamine intermediate as a β-lactamase inhibition strategy probed in inhibitor-resistant SHV β-lactamase variants.

Ke Wei W Rodkey Elizabeth A EA Sampson Jared M JM Skalweit Marion J MJ Sheri Anjaneyulu A Pagadala Sundar Ram Reddy SR Nottingham Michael D MD Buynak John D JD Bonomo Robert A RA van den Akker Focco F

ChemMedChem 20120321 6

The ability of bacteria to express inhibitor-resistant (IR) β-lactamases is stimulating the development of novel inhibitors of these enzymes. The 2'β-glutaroxypenicillinate sulfone, SA2-13, was previously designed to enhance the stabilization of the deacylation-refractory, trans-enamine inhibitory intermediate. To test whether this mode of inhibition can overcome different IR mutations, we determined the binding mode of SA2-13 through X-ray crystallography, obtaining co-crystals of the inhibitor ...[more]

PMID: 22438274

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Project description:The objective of this study was to determine the molecular factors that lead to beta-lactamase inhibitor resistance for the M69V variant in SHV-1 beta-lactamase. With mechanism-based inhibitors, the beta-lactamase forms an acyl-enzyme intermediate that consists of a trans-enamine derivative in the active site. This study focuses on these intermediates by introducing the E166A mutation that greatly retards deacylation. Thus, by comparing the properties of the E166A and M69V/E166A forms, we can explore the consequences of the resistance mutation at the level of the enamine acyl-enzyme forms. The reactions between the beta-lactamase and the inhibitors tazobactam, sulbactam, and clavulanic acid are followed in single crystals of the enzymes by using a Raman microscope. The resulting Raman difference spectroscopic data provide detailed information about conformational events involving the enamine species as well as an estimate of their populations. The Raman difference spectra for each of the inhibitors in the E166A and M69V/E166A variants are very similar. In particular, detailed analysis of the main enamine Raman vibration near 1595 cm(-1) reveals that the structure and flexibility of the enamine fragments are essentially identical for each of the three inhibitors in E166A and in the M69V/E166A double mutant. This finding is in accord with the X-ray-derived structures, presented herein at 1.6-1.75 A resolution, of the trans-enamine intermediates formed by the three inhibitors in M69V/E166A. However, a comparison of Raman results for M69V/E166A and E166A shows that the M69V mutation results in a 40%, 25%, and negligible reductions in the enamine population when the beta-lactamase crystals are soaked in 5 mM tazobactam, clavulanic acid, and sulbactam solutions, respectively. The levels of enamine from tazobactam and clavulanic acid can be increased by increasing the concentrations of inhibitor in the mother liquor. Thus, the sensitivity of population levels to the inhibitor concentration in the mother liquor focuses attention on the properties of the encounter complex preceding acylation. It is proposed that for small ligands, such as tazobactam, sulbactam, and clavulanic acid, the positioning of the lactam ring in the active site in the correct orientation for acylation is only one of a number of poorly defined conformations. For tazobactam and clavulanic acid, the correctly oriented encounter complex is even less likely in the M69V variant, leading to a reduction in the level of inhibition of the enzyme via formation of the acyl-enzyme intermediate and the onset of resistance. Analysis of the X-ray structures of the three intermediates in M69V/E166A demonstrates that, compared to the structures for the E166A form, the oxyanion hole becomes smaller, providing one explanation for why acylation may be less efficient following the M69V substitution.

Project description:?-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of ?-lactam antibiotics to treat infections. A novel non-?-lactam ?-lactamase inhibitor with activity against many class A and C and some class D ?-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 ?-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 ?-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 ?-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 ?-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 ?-lactamases, S130 may be the most important amino acid for the inhibition of class A ?-lactamases, perhaps even for the novel diazabicyclooctane class of ?-lactamase inhibitors.

Project description:Beta-lactamases are enzymes that catalyze the hydrolysis of beta-lactam antibiotics. beta-lactamase/beta-lactamase inhibitor protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein-protein interactions. BLIP is a 165 amino acid protein that inhibits several class A beta-lactamases with a wide range of affinities: picomolar affinity for K1; nanomolar affinity for TEM-1, SME-1, and BlaI; but only micromolar affinity for SHV-1 beta-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high-resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of computational design methodology. We used EGAD, a physics-based computational design program, to redesign BLIP in an attempt to increase affinity for SHV-1. Characterization of several of designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10- to 1000-fold relative to wild type BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of the calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Structures of the two complexes with the highest affinity, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 A resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in the experimental structures, but not in the predicted design conformations. This discrepancy highlights the difficulty of modeling salt bridge interactions with a protein design algorithm that approximates side chains as discrete rotamers. Nevertheless, while local structural features of the interface were sometimes miscalculated, EGAD is globally successful in designing complexes with increased affinity.

Dataset Information

The importance of the trans-enamine intermediate as a ?-lactamase inhibition strategy probed in inhibitor-resistant SHV ?-lactamase variants.

Publications

The importance of the trans-enamine intermediate as a β-lactamase inhibition strategy probed in inhibitor-resistant SHV β-lactamase variants.

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