AUTHOR=Thakur Naveen , Sharma Amar Nath , Hade Mangesh Dattu , Chhaya Ajay , Kumar Ashwani , Jolly Ravinder Singh , Dikshit Kanak L. 

TITLE=New Insights Into the Function of Flavohemoglobin in Mycobacterium tuberculosis: Role as a NADPH-Dependent Disulfide Reductase and D-Lactate-Dependent Mycothione Reductase

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=11

YEAR=2022

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2021.796727

DOI=10.3389/fcimb.2021.796727

ISSN=2235-2988

ABSTRACT=<p><italic>Mycobacterium tuberculosis</italic> (<italic>Mtb</italic>) produces an unconventional flavohemoglobin (<italic>Mtb</italic>FHb) that carries a FAD-binding site similar to D-lactate dehydrogenases (D-LDH) and oxidizes D-lactate into pyruvate. The molecular mechanism by which <italic>Mtb</italic>FHb functions in <italic>Mtb</italic> remains unknown. We discovered that the D-LDH-type FAD-binding site in <italic>Mtb</italic>FHb overlaps with another FAD-binding motif similar to thioredoxin reductases and reduces DTNB in the presence of NADPH similar to trxB of <italic>Mtb</italic>. These results suggested that <italic>Mtb</italic>FHb is functioning as a disulfide oxidoreductase. Interestingly, D-lactate created a conformational change in <italic>Mtb</italic>FHb and attenuated its ability to oxidize NADPH. Mass spectroscopy demonstrated that <italic>Mtb</italic>FHb reduces des-<italic>myo-</italic>inositol mycothiol in the presence of D-lactate unlike NADPH, indicating that D-lactate changes the specificity of <italic>Mtb</italic>FHb from di-thiol to di-mycothiol. When <italic>M. smegmatis</italic> carrying deletion in the <italic>fhb</italic>II gene (encoding a homolog of <italic>Mtb</italic>FHb) was complemented with the <italic>fhb</italic> gene of <italic>Mtb</italic>, it exhibited four- to fivefold reductions in lipid peroxidation and significant enhancement in the cell survival under oxidative stress. These results were corroborated by reduced lipid peroxidation and enhanced cell survival of wild-type <italic>M. smegmatis</italic> after overexpression of the <italic>fhb</italic> gene of <italic>Mtb</italic>. Since D-lactate is a by-product of lipid peroxidation and <italic>Mtb</italic>FHb is a membrane-associated protein, D-lactate-mediated reduction of mycothiol disulfide by <italic>Mtb</italic>FHb may uniquely equip <italic>Mtb</italic> to relieve the toxicity of D-lactate accumulation and protect the cell from oxidative damage, simultaneously balancing the redox environment under oxidative stress that may be vital for the pathogenesis of <italic>Mtb</italic>.</p>