Dataset Information

Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam.

ABSTRACT: For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.

SUBMITTER: Malla TN

PROVIDER: S-EPMC10485065 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Publications

Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam.

Malla Tek Narsingh TN Zielinski Kara K Aldama Luis L Bajt Sasa S Feliz Denisse D Hayes Brendon B Hunter Mark M Kupitz Christopher C Lisova Stella S Knoska Juraj J Martin-Garcia Jose Manuel JM Mariani Valerio V Pandey Suraj S Poudyal Ishwor I Sierra Raymond G RG Tolstikova Alexandra A Yefanov Oleksandr O Yoon Chung Hong CH Ourmazd Abbas A Fromme Petra P Schwander Peter P Barty Anton A Chapman Henry N HN Stojkovic Emina A EA Batyuk Alexander A Boutet Sébastien S Phillips George N GN Pollack Lois L Schmidt Marius M

Nature communications 20230907 1

For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crysta ...[more]

PMID: 37679343

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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

Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam.

Publications

Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam.

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