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Menaquinone (Vitamin K2) plays a vital role in energy generation and environmental adaptation in many bacteria, includingMycobacterium tuberculosis(Mtb). Although menaquinone levels are known to be tightly linked to the redox/energy status of the cell, the regulatory mechanisms underpinning this phenomenon are unclear. The first committed step in menaquinone biosynthesis is catalyzed by MenD, a thiamine diphosphate-dependent enzyme comprising three domains. Domains I and III form the MenD active site, but no function has yet been ascribed to domain II. Here we show the last cytosolicmetabolite in the menaquinone biosynthesis pathway (1,4-dihydroxy-2-napthoic acid, DHNA) binds to domain II ofMtb-MenD and inhibits enzyme activity. We identified three arginine residues (Arg97, Arg277 and Arg303) that are important for both enzyme activity and the feedback inhibition by DHNA: Arg277 appears to be particularly important for signal propagation from the allosteric site to the active site. This is the first evidence of feedback regulation of the menaquinone biosynthesis pathway in bacteria, unravelling a protein level regulatory mechanism for control of menaquinone levels within the cell.

Cofactor F420plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420has not been fully eluci-dated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we show that the guanylyltransferases FbiD and CofC accept phosphoenolpyruvate, rather than 2-phospho-L-lactate, as their substrate, leading to the formation of the previously uncharacterized intermediate, dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420species when combined with the γ-glutamyl ligase activity of the N-terminal domain. This new insight has allowed the heterologous expression F420from a recombinant F420biosynthetic pathway inEscherichia coli.