4MBK: Crystal structure of K234R inhibitor-resistant variant of SHV beta-lactamase in complex with SA2-13

Citation:
Abstract
beta-Lactamases are the major reason beta-lactam resistance is seen in Gram-negative bacteria. To combat this resistance mechanism, beta-lactamase inhibitors are currently being developed. Presently, there are only three that are in clinical use (clavulanate, sulbactam and tazobactam). In order to address this important medical need, we explored a new inhibition strategy that takes advantage of a long-lived inhibitory trans-enamine intermediate. SA2-13 was previously synthesized and shown to have a lower k(react) than tazobactam. We investigated here the importance of the carboxyl linker length and composition by synthesizing three analogs of SA2-13 (PSR-4-157, PSR-4-155, and PSR-3-226). All SA2-13 analogs yielded higher turnover numbers and k(react) compared to SA2-13. We next demonstrated using protein crystallography that increasing the linker length by one carbon allowed for better capture of a trans-enamine intermediate; in contrast, this trans-enamine intermediate did not occur when the C2 linker length was decreased by one carbon. If the linker was altered by both shortening it and changing the carboxyl moiety into a neutral amide moiety, the stable trans-enamine intermediate in wt SHV-1 did not form; this intermediate could only be observed when a deacylation deficient E166A variant was studied. We subsequently studied SA2-13 against a relatively recently discovered inhibitor-resistant (IR) variant of SHV-1, SHV K234R. Despite the alteration in the mechanism of resistance due to the K-->R change in this variant, SA2-13 was effective at inhibiting this IR enzyme and formed a trans-enamine inhibitory intermediate similar to the intermediate seen in the wt SHV-1 structure. Taken together, our data reveals that the C2 side chain linker length and composition profoundly affect the formation of the trans-enamine intermediate of penam sulfones. We also show that the design of SA2-13 derivatives offers promise against IR SHV beta-lactamases that possess the K234R substitution.
PDB ID: 4MBKDownload
MMDB ID: 121903
PDB Deposition Date: 2013/8/19
Updated in MMDB: 2017/11
Experimental Method:
x-ray diffraction
Resolution: 1.46  Å
Source Organism:
Similar Structures:
Biological Unit for 4MBK: monomeric; determined by author and by software (PISA)
Molecular Components in 4MBK
Label Count Molecule
Protein (1 molecule)
1
Beta-lactamase Shv-1
Molecule annotation
Chemicals (3 molecules)
1
2
2
1
* Click molecule labels to explore molecular sequence information.

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