1. Biochemistry and Chemical Biology
  2. Computational and Systems Biology
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Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase

  1. John Z Chen
  2. Douglas M Fowler
  3. Nobuhiko Tokuriki  Is a corresponding author
  1. University of British Columbia, Canada
  2. University of Washington, United States
Research Article
  • Cited 4
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Cite this article as: eLife 2020;9:e56707 doi: 10.7554/eLife.56707

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 (25oC and 37oC). 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

Article and author information

Author details

  1. John Z Chen

    Michael Smith Laboratories, University of British Columbia, Vancouver, Canada
    Competing interests
    The authors declare that no competing interests exist.
  2. Douglas M Fowler

    Genome Sciences, Bioengineering, University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7614-1713
  3. 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.
    ORCID icon "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.

Reviewing Editor

  1. Sarel Jacob Fleishman, Weizmann Institute of Science, Israel

Publication history

  1. Received: March 6, 2020
  2. Accepted: June 6, 2020
  3. Accepted Manuscript published: June 8, 2020 (version 1)
  4. Version of Record published: June 22, 2020 (version 2)

Copyright

© 2020, Chen et al.

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.

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