Abstract
Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial for maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely, those from S. aureus and B. cereus. Our analyses reveal a uniquely organized biological tetramer; however, the structure of the monomeric subunit is highly similar to those of other flavoprotein disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in the proximity of the FAD cofactor. We also report enzymatic activities for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.
Highlights
Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections
We report enzymatic activities for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria
GSH is continuously oxidized to glutathione disulfide (GSSG), which can be rapidly converted back to GSH by glutathione reductase (GR),[3] to maintain the required GSH/GSSG ratio that is important for the cellular redox balance
Summary
Mycothiol; BSH, bacillithiol; BSSB, bacillithiol disulfide; NMe-BSH, N-methyl-bacillithiol; Bc, Bacillus cereus; Bs, Bacillus subtilis; Ba, Bacillus anthracis; Sa, Staphylococcus aureus; GR, glutathione reductase; TrxR, thioredoxin reductase; FPMO, Experimental procedures used in this study, including protein expression, purification, and characterization; activity measurements; protein crystallography; and bioinformatics and structural analysis (Figures S1−S14 and Tables S1−S3) (PDF). Flavoprotein monooxygenase; FNR, ferredoxin/flavodoxin NAD(P)+ oxidoreductase; Fld, flavodoxin; Grx, glutaredoxin; Brx, bacilliredoxin; BshA/B/C, BSH biosynthesis enzymes A/ B/C; YpdA, bacillithiol disulfide reductase Bdr; tDBDF, twodinucleotide binding domain flavoprotein superfamily; FAD, flavin adenine dinucleotide; NADPH, nicotinamide adenine. ■ AUTHOR INFORMATION adenine dinucleotide phosphate, oxidized form; PAGE, polyacrylamide gel electrophoresis; DLS, dynamic light scattering; E0,́ standard reduction potential at pH 7
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