Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus

Roberts, Alexandra A., Sharma, Sunil V., Strankman, Andrew W., Duran, Shayla R., Rawat, Mamta, and Hamilton, Chris J. (2013) Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus. Biochemical Journal, 451 (1). pp. 69-79.

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Abstract

FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G+C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The Km values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn2+ and Mg2+ are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS-fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.

Item ID: 25722
Item Type: Article (Refereed Research - C1)
Keywords: antibiotic detoxification, bacillithiol, metalloenzyme, substrate mimic
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ISSN: 1470-8728
Funders: Biotechnology and Biological Sciences Research Council, National Institutes of Health (NIH)
Projects and Grants: BB/H013504/1, 1SC3GM100855
Date Deposited: 03 May 2013 05:40
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060107 Enzymes @ 70%
06 BIOLOGICAL SCIENCES > 0605 Microbiology > 060501 Bacteriology @ 10%
06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060109 Proteomics and Intermolecular Interactions (excl Medical Proteomics) @ 20%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 60%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 40%
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