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

Abstract

Bacillithiol is a glucosamine‐derived antioxidant found in several pathogenic Gram‐positive bacteria. The compound is involved in maintaining the appropriate redox state within the cell as well as detoxifying foreign agents like the antibiotic fosfomycin. Bacillithiol is produced via the action of three enzymes, including BshA, a retaining GT‐B glycosyltransferase that utilizes UDP‐N‐acetylglucosamine and l‐malate to produce N‐acetylglucosaminyl‐malate. Recent studies suggest that retaining GT‐B glycosyltransferases like BshA utilize a substrate‐assisted mechanism that goes through an SNi‐like transition state. In a previous study, we relied on X‐ray crystallography as well as computational simulations to hypothesize the manner in which substrates would bind the enzyme, but several questions about substrate binding and the role of one of the amino acid residues persisted. Another study demonstrated that BshA might be subject to feedback inhibition by bacillithiol, but this phenomenon was not analyzed further to determine the exact mechanism of inhibition. Here we present X‐ray crystallographic structures and steady‐state kinetics results that help elucidate both of these issues. Our ligand‐bound crystal structures demonstrate that the active site provides an appropriate steric and geometric arrangement of ligands to facilitate the substrate‐assisted mechanism. Finally, we show that bacillithiol is competitive for UDP‐N‐acetylglucosamine with a Ki value near 120–130 μM and likely binds within the BshA active site, suggesting that bacillithiol modulates BshA activity via feedback inhibition. The work presented here furthers our understanding of bacillithiol metabolism and can aid in the development of inhibitors to counteract resistance to antibiotics such as fosfomycin.

<em>In silico</em> study of thiophenic heterocycles against <em>Staphylococcus aureus</em> targets

“…Moreover, molecular docking was performed using the software Molegro Virtual Docker 6.0 [ 5 ] to calculate the energies of total ligand-receptor interactions with all chosen proteins (PDB ID 5ZNJ [ 6 ] and PDB ID 6N1X [ 7 ]), rank the thiophenics tested with some bactericidal drugs used as controls in this study and analyze the types of interactions involved between the molecules and the active sites of each protein used.…”

Towards Computationally Guided Design and Engineering of a Neisseria Meningitidis Serogroup W Capsule Polymerase with Altered Substrate Specificity

“…Among these nine residues, 4 are identical between the two proteins (H144, V170, E307, and F309) (Figure 4). It has been shown in other published studies that mutation of NmW GT E307A leads to the complete abolishment of galactosyltransferase activity in that domain [19]. In addition to the likely effects due the stereochemical differences of glucose and galactose moieties at carbon 4, the residues in proximity to the N-acetyl group (R234 and S306) are not conserved between the two proteins and are good targets for modification.…”

“…The rotatable bonds notwithstanding, the differences in the hexose moieties of interest are located at 2 carbon atoms, C2, which carries an -OH or N-acetyl group and C4, where the difference is in the orientation of the -OH group. Analysis of protein-ligand interactions was based on the crystal structures of BshA bound to UDP and with UDP and N-acetylglucosamine (GlcNAc) as reported in the literature [19]. NmW GT differs in substrate specificity in that it utilizes UDP-Gal and these studies seek to change the specificity of this domain to UDP-GalNAc (Figure 3).…”

“…The structures of several members of the BshA family, also classified as GT4 family enzymes, are available. These include family members from pathogenic and non-pathogenic species such as Bacillus subtilis, Bacillus anthracis, Staphylococcus epidermis, and Staphylococcus aureus [18][19][20]. The crystal structures reveal the active site and interactions between the UDP and hexose moieties with specific amino acid residues.…”

A Glycolipid Glycosyltransferase with Broad Substrate Specificity from the Marine Bacterium “ Candidatus Pelagibacter sp.” Strain HTCC7211

“…In contrast with GT cp , the Pgts from M. loti and A. tumefaciens favour uridine UDP-Gal over UDP-Glc(15,16). A comparison of GT cp kinetics with other biochemically characterized GT4 glycosyltransferases is listed in Table4(22,(28)(29)(30)(31)(32)(33).The effect of various metal ions on the enzyme activity of GT cp is shown in Fig.4F. Among the tested metal ions (5 mM), Mg 2+ , Ca 2+ , and Mn 2+ significantly stimulated GT cp activity by up to 223%, 125%, and 138%, respectively, although the as-isolated enzyme is already active Furthermore, EDTA did not significantly affect the enzymatic activity of GT cp after incubation for 60 min at room temperature.…”