Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase

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Abstract

Metallo-β-lactamases (MBLs) degrade a broad spectrum of β-lactam antibiotics, and are a major disseminating source for multidrug resistant bacteria. Despite many biochemical studies in diverse MBLs, molecular understanding of the roles of residues in the enzyme's stability and function, and especially substrate specificity, is lacking. Here, we employ deep mutational scanning (DMS) to generate comprehensive single amino acid variant data on a major clinical MBL, VIM-2, by measuring the effect of thousands of VIM-2 mutants on the degradation of three representative classes of β-lactams (ampicillin, cefotaxime, and meropenem) and at two different temperatures (25^oC and 37^oC). We revealed residues responsible for expression and translocation, and mutations that increase resistance and/or alter substrate specificity. The distribution of specificity-altering mutations unveiled distinct molecular recognition of the three substrates. Moreover, these function-altering mutations are frequently observed among naturally occurring variants, suggesting that the enzymes have continuously evolved to become more potent resistance genes.

Data availability

All processed data that are analyzed are included in the manuscript and supporting files. Raw sequencing data has been deposited in the NCBI Sequencing Read Archive and all files are submitted under the BioProject accession code PRJNA606894.

The following data sets were generated

(2020) VIM-2 deep mutational scanning
NCBI Sequencing Read Archive, PRJNA606894.

https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA606894

Article and author information

Author details

John Z Chen

Michael Smith Laboratories, University of British Columbia, Vancouver, Canada

Competing interests
The authors declare that no competing interests exist.
Douglas M Fowler

Genome Sciences, Bioengineering, University of Washington, Seattle, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7614-1713
Nobuhiko Tokuriki

Michael Smith Laboratory, University of British Columbia, Vancouver, Canada

For correspondence
tokuriki@msl.ubc.ca

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8235-1829

Funding

Canadian Institute of Health Research (FDN-148437)

Nobuhiko Tokuriki

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.