Dataset Information

Structures of SHV-1 ?-lactamase with penem and penam sulfone inhibitors that form cyclic intermediates stabilized by carbonyl conjugation.

ABSTRACT: Bacterial ?-lactamase enzymes are in large part responsible for the decreased ability of ?-lactam antibiotics to combat infections. The inability to overcome ?-lactamase mediated resistance spurred the development of inhibitors with penems and penam sulfones being amongst the most potent and broad spectrum mechanism-based inactivators. These inhibitors form covalent, "suicide-type" inhibitory intermediates that are attached to the catalytic S70 residue. To further probe the details of the mechanism of ?-lactamase inhibition by these novel compounds, we determined the crystal structures of SHV-1 bound with penem 1, and penam sulfones SA1-204 and SA3-53. Comparison with each other and with previously determined crystal structures of members of these classes of inhibitors suggests that the final conformation of the covalent adduct can vary greatly amongst the complex structures. In contrast, a common theme of carbonyl conjugation as a mechanism to avoid deacylation emerges despite that the penem and penam sulfone inhibitors form different types of intermediates. The detailed insights gained from this study could be used to further improve new mechanism-based inhibitors of these common class A serine ?-lactamases.

SUBMITTER: Ke W

PROVIDER: S-EPMC3493512 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Structures of SHV-1 β-lactamase with penem and penam sulfone inhibitors that form cyclic intermediates stabilized by carbonyl conjugation.

Ke Wei W Pattanaik Priyaranjan P Bethel Christopher R CR Sheri Anjaneyulu A Buynak John D JD Bonomo Robert A RA van den Akker Focco F

PloS one 20121108 11

Bacterial β-lactamase enzymes are in large part responsible for the decreased ability of β-lactam antibiotics to combat infections. The inability to overcome β-lactamase mediated resistance spurred the development of inhibitors with penems and penam sulfones being amongst the most potent and broad spectrum mechanism-based inactivators. These inhibitors form covalent, "suicide-type" inhibitory intermediates that are attached to the catalytic S70 residue. To further probe the details of the mechan ...[more]

PMID: 23145056

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Project description:The reactions between single crystals of the SHV-1 beta-lactamase enzyme and the carbapenems, meropenem, imipenem, and ertapenem, have been studied by Raman microscopy. Aided by quantum mechanical calculations, major populations of two acyl-enzyme species, a labile Delta (2)-pyrroline and a more tightly bound Delta (1)-pyrroline, have been identified for all three compounds. These isomers differ only in the position of the double bond about the carbapenem nucleus. This discovery is consonant with X-ray crystallographic findings that also identified two populations for meropenem bound in SHV-1: one with the acyl CO group in the oxyanion hole and the second with the acyl group rotated 180 degrees compared to its expected position [Nukaga, M., Bethel, C. R., Thomson, J. M., Hujer, A. M., Distler, A. M., Anderson, V. E., Knox, J. R., and Bonomo, R. A. (2008) J. Am. Chem. Soc. (in press)]. When crystals of the Delta (1)- and Delta (2)-containing acyl-enzymes were exposed to solutions with no carbapenem, rapid deacylation of the Delta (2) species was observed by kinetic Raman experiments. However, no change in the Delta (1) population was observed over 1 h, the effective lifetime of the crystal. These observations lead to the hypothesis that the stable Delta (1) species is due to the form seen by X-ray with the acyl carbonyl outside the oxyanion hole, while the Delta (2) species corresponds to the form with the carbonyl inside the oxyanion hole. Soak-in and soak-out Raman experiments also demonstrated that tautomeric exchange between the Delta (1) and Delta (2) forms does not occur on the crystalline enzyme. When meropenem or ertapenem was reacted with SHV-1 in solution, the Raman difference spectra demonstrated that only a major population corresponding to the Delta (1) acyl-enzyme could be detected. The 1003 cm (-1) mode of the phenyl ring positioned on the C3 side chain of ertapenem acts as an effective internal Raman intensity standard, and the ratio of its intensity to that of the 1600 cm (-1) feature of Delta (1) provides an estimate of the relative populations of Delta (1). In solution, I 1600/ I 1003 equals 2, and in the crystal, I 1600 /I 1003 equals 1. This is strong evidence that the Delta (1) and Delta (2) acyl-enzymes in the crystal are present in approximately equal amounts, in agreement with the X-ray data. However, in solution there are twice as many Delta (1) species per Phe group, and this represents approximately 100% of the active sites, which is consistent with the observed inhibition of the enzyme's activity.

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:Among the TEM-type extended-spectrum beta-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV beta-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in beta-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys beta-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser beta-lactamases. Correspondingly, the overall catalytic efficiency (kcat/Km) of hydrolysis for cefotaxime was increased from 0.60 +/- 0.07 microM(-1) s(-1) (mean +/- standard deviation) for Gly238Ser to 1.70 +/- 0.01 microM(-1) s(-1) for the Asp104Lys and Gly238Ser beta-lactamase (threefold increase). We also showed that (i) k3 was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the Km for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser beta-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 beta-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.

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

Structures of SHV-1 ?-lactamase with penem and penam sulfone inhibitors that form cyclic intermediates stabilized by carbonyl conjugation.

Publications

Structures of SHV-1 β-lactamase with penem and penam sulfone inhibitors that form cyclic intermediates stabilized by carbonyl conjugation.

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