A Structural, Functional, and Computational Analysis of BshA, the First Enzyme in the Bacillithiol Biosynthesis Pathway

Abstract

Bacillithiol is a compound produced by several Gram-positive bacterial species including the human pathogens Staphylococcus aureus and Bacillus anthracis. It is involved in maintaining cellular redox balance as well as the destruction of reactive oxygen species and harmful xenobiotic agents, including the antibiotic fosfomycin. BshA, BshB, and BshC are the enzymes involved in bacillithiol biosynthesis. BshA is a retaining glycosyltransferase responsible for the first committed step in bacillithiol production, namely the addition of N-acetylglucosamine to l-malate. Retaining glycosyltransferases like BshA are proposed to utilize an SNi-like reaction mechanism in which leaving group departure and nucleophilic attack occur on the same face of the hexose. However, significant questions persist regarding the details of how BshA and similar enzymes accommodate their substrates and facilitate catalysis. Here we report X-ray crystallographic structures of BshA from Bacillus subtilis 168 bound with UMP and/or GlcNAc-mal at resolutions of 2.15 and 2.02 Å, respectively. These ligand-bound structures, along with our functional and computational studies, provide clearer insight into how BshA and other retaining GT-B glycosyltransferases operate, corroborating the substrate-assisted, SNi-like reaction mechanism. The analyses presented herein can serve as the basis for the design of inhibitors capable of preventing bacillithiol production and subsequently, help combat resistance to fosfomycin in various pathogenic Gram-positive microorganisms.

A structural and functional analysis of the glycosyltransferase BshA from Staphylococcus aureus: Insights into the reaction mechanism and regulation of bacillithiol production

“…S1, Panel A). This obligatory homodimer was shown to be the native form of B. subtilis BshA . Examination of the unit cell contents in this study shows that this interface is indeed present in the S. aureus BshA enzyme.…”

“…This obligatory homodimer was shown to be the native form of B. subtilis BshA. 11 Examination of the unit cell contents in this study shows that this interface is indeed present in the S. aureus BshA enzyme. Analysis by the proteins, interfaces, structures, and assemblies (PISA) tool at the PDBePISA 14 website indicates that~5900 Å 2 of surface area is buried within this dimeric interface with a ΔG of formation of −44 kcal/mol.…”

“…Data were processed and scaled within the AutoPROC program (BshA/UDP complex) or with HKL2000 (BshA/UDP + N ‐acetylglucosamine complex). The structure of S. aureus BshA with UDP bound was solved via molecular replacement with Phaser using a Phyre2 model based on the structure of BshA from B. subtilis [PDB entry http://firstglance.jmol.org/fg.htm?mol=5D01] as the search ensemble. Manual model adjustments with Coot and refinement with Phenix produced a final model with R overall and R free values of 16.9% and 18.8%, respectively.…”

“…These enzymes indeed display sequential rather than ping‐pong kinetics, which indicates a direct reaction between the glycosyl donor and acceptor, but we sought structural evidence to support such a mechanism. Previously, we determined the X‐ray crystallographic structure of product‐bound BshA enzyme from Bacillus subtilis . Suitable crystals of this ortholog grew only in the presence of both substrates, and the N ‐acetylglucosaminyl‐malate product was always observed in the active site.…”

“…Previously, we determined the X-ray crystallographic structure of product-bound BshA enzyme from Bacillus subtilis. 11 Suitable crystals of this ortholog grew only in the presence of both substrates, and the N-acetylglucosaminylmalate product was always observed in the active site. While we were able to make several conclusions about the enzyme's function, our analysis was limited in part by our inability to observe the substrate UDP-Nacetylglucosamine in the active site by itself.…”