DprE2 is a molecular target of the anti-tubercular nitroimidazole compounds pretomanid and delamanid

Abstract Mycobacterium tuberculosis is one of the global leading causes of death due to a single infectious agent. Pretomanid and delamanid are new antitubercular agents that have progressed through the drug discovery pipeline. These compounds are bicyclic nitroimidazoles that act as pro-drugs, requiring activation by a mycobacterial enzyme; however, the precise mechanisms of action of the active metabolite(s) are unclear. Here, we identify a molecular target of activated pretomanid and delamanid: the DprE2 subunit of decaprenylphosphoribose-2’-epimerase, an enzyme required for the synthesis of cell wall arabinogalactan. We also provide evidence for an NAD-adduct as the active metabolite of pretomanid. Our results highlight DprE2 as a potential antimycobacterial target and provide a foundation for future exploration into the active metabolites and clinical development of pretomanid and delamanid

Tat–Dependent Translocation of an F420–Binding Protein of Mycobacterium tuberculosis

F420 is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F420 has an important role in the oxidoreductive reactions of Mycobacterium tuberculosis, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N–terminal signal sequence and is annotated as an F420–dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co–purify with F420 during expression experiments in M. smegmatis. We also show that the Rv0132c–F420 complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F420–binding protein being a substrate for the Tat pathway and of the presence of F420 outside of the cytosol in any F420–producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the Mycobacterium tuberculosis complex. Taken together, these results show that current understanding of F420 biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope

A revised biosynthetic pathway for the cofactor F-420 in prokaryotes

Cofactor F420 plays 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 F420 has not been fully elucidated: 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 present the structure of the guanylyltransferase FbiD and show that, along with its archaeal homolog CofC, it accepts phosphoenolpyruvate, rather than 2-phospho-L-lactate, as the 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 F420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F420 from a recombinant F420 biosynthetic pathway in Escherichia coli

Metabolic Engineering of Cofactor F420 Production in Mycobacterium smegmatis

Cofactor F420 is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F420 has a direct and important role in archaeal energy metabolism whereas the role of F420 in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F420 has a role in the pathogenesis of M. tuberculosis, the causative agent of tuberculosis. In the absence of a commercial source for F420, M. smegmatis has previously been used to provide this cofactor for studies of the F420-dependent proteins from mycobacterial species. Three proteins have been shown to be involved in the F420 biosynthesis in Mycobacteria and three other proteins have been demonstrated to be involved in F420 metabolism. Here we report the over-expression of all of these proteins in M. smegmatis and testing of their importance for F420 production. The results indicate that co–expression of the F420 biosynthetic proteins can give rise to a much higher F420 production level. This was achieved by designing and preparing a new T7 promoter–based co-expression shuttle vector. A combination of co–expression of the F420 biosynthetic proteins and fine-tuning of the culture media has enabled us to achieve F420 production levels of up to 10 times higher compared with the wild type M. smegmatis strain. The high levels of the F420 produced in this study provide a suitable source of this cofactor for studies of F420-dependent proteins from other microorganisms and for possible biotechnological applications

Cloning, expression, purification, crystallization and preliminary X-ray studies of the C-terminal domain of Rv3262 (FbiB) from Mycobacterium tuberculosis

During cofactor F420 biosynthesis, the enzyme F420-[gamma]-glutamyl ligase (FbiB) catalyzes the addition of [gamma]-linked L-glutamate residues to form polyglutamylated F420 derivatives. In Mycobacterium tuberculosis, Rv3262 (FbiB) consists of two domains: an N-terminal domain from the F420 ligase superfamily and a C-terminal domain with sequence similarity to nitro-FMN reductase superfamily proteins. To characterize the role of the C-terminal domain of FbiB in polyglutamyl ligation, it has been purified and crystallized in an apo form. The crystals diffracted to 2.0 Å resolution using a synchrotron source and belonged to the tetragonal space group P41212 (or P43212), with unit-cell parameters a = b = 136.6, c = 101.7 Å, [alpha] = [beta] = [gamma] = 90°

Elongation of the poly-γ-glutamate tail of F420 requires both domains of the F420:γ-glutamyl ligase (FbiB) of Mycobacterium tuberculosis

Cofactor F420 is an electron carrier with a major role in the oxidoreductive reactions of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). A γ-glutamyl ligase catalyzes the final steps of the F420 biosynthesis pathway by successive additions of L-glutamate residues to F420-0, producing a poly-γ-glutamate tail. The enzyme responsible for this reaction in Archaea (CofE) comprises a single domain and produces F420-2 as the major species. The homologous Mtb enzyme, FbiB, is a two-domain protein and produces F420 with predominantly 5-7 L-glutamate residues in the poly-γ-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated γ-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here we demonstrate that full-length FbiB adds multiple L-glutamate residues to F420-0 in vitro to produce F420-5 after 24 hours; communication between the two domains is critical for full γ-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo, F420-0 and FMN bound states, displaying distinct sites for F420-0 and FMN ligands that partially overlap. Finally, we discuss the features of a full-length structural model produced by small angle X-ray scattering (SAXS) and its implications for the role of N- and C-terminal domains in catalysis

Investigating the Reaction Mechanism of F₄₂₀-Dependent Glucose-6-phosphate Dehydrogenase from <i>Mycobacterium tuberculosis</i>: Kinetic Analysis of the Wild-Type and Mutant Enzymes

F₄₂₀-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using F₄₂₀ cofactor as the hydride transfer acceptor, within mycobacteria. A previous crystal structure of wild-type FGD led to a proposed mechanism suggesting that the active site residues His40, Trp44, and Glu109 could be involved in catalysis. We have characterized the wild-type FGD and five FGD variants (H40A, W44F, W44Y, W44A, and E109Q) by fluorescence binding assays and steady-state and pre-steady-state kinetic experiments. Compared to wild-type FGD, all the variants had lower binding affinities for F₄₂₀, thus suggesting that Trp44, His40, and Glu109 aid in F₄₂₀ binding. While all the variants had decreased catalytic efficiencies, FGD H40A and W44A were the least efficient, having lost ∼1000- and ∼2000-fold activity, respectively. This confirms a crucial catalytic role for His40 in the FGD reaction and suggests that aromaticity at residue 44 aids catalysis. To investigate the proposed roles of Glu109 and His40 in acid–base catalysis, the pH dependence of kinetic parameters has been determined for the E109Q and H40A mutants and compared to those of the wild-type enzyme. The log <i>k</i>_cat–pH profile of wild-type FGD and E109Q revealed two ionizable residues in the enzyme–substrate complex, while H40A displayed only one ionization event. The FGD E109Q variant displayed pH-dependent kinetic cooperativity with respect to the F₄₂₀ cofactor. The multiple-turnover pre-steady-state kinetics were biphasic for wild-type FGD, W44F, W44Y, and E109Q, while the H40A and W44A variants displayed only a single phase because of their reduced catalytic efficiency