Abstact

The role of the cell envelope-associated Rv0132c/FGD2 from Mycobacterium tuberculosis has long been a subject of debate. Importantly, FGD2 is found only in pathogenic mycobacteria, making it a potential drug target. While some suggest it functions as a glucose-6-phosphate dehydrogenase, others propose it acts instead as an F(420)-dependent hydroxy-mycolic acid dehydrogenase-an activity linked to cell-wall remodeling and inhibition by the anti-tubercular drug pretomanid. Yet, direct evidence for either activity has been lacking. Here, we heterologously express and purify active Mtb-FGD2, and demonstrate that the enzyme binds the F(420) cofactor with nanomolar affinity. Crystal structures for both the apo-form and the F(420) complex reveal that the Mtb-FGD2 active site architecture is consistent with sugar substrates but notably lacks a phosphate-binding pocket. Biochemical assays confirm that Mtb-FGD2 functions efficiently as an F(420)-dependent glucose dehydrogenase in vitro. Computational docking combined with molecular dynamics simulations further supports the formation of a catalytically plausible beta-D-glucose:F(420) ternary complex. When coupled to other F(420)-dependent enzymes, Mtb-FGD2 readily supports glucose-driven F(420).H(2)-dependent oxidoreductase activity. Our data thus suggest that the Mtb-FGD2 provides reduced F(420).H(2) in a glucose-dependent manner to support mycobacterial F(420).H(2)-dependent oxidoreductases in the cell envelope.