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

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

Cofactor F420, an emerging redox power in biosynthesis of secondary metabolites

Cofactor F420 is a low-potential hydride-transfer deazaflavin that mediates important oxidoreductive reactions in the primary metabolism of archaea and a wide range of bacteria. Over the past decade, biochemical studies have demonstrated another essential role for F420 in the biosynthesis of various classes of natural products. These studies have substantiated reports predating the structural determination of F420 that suggested a potential role for F420 in the biosynthesis of several antibiotics produced by Streptomyces. In this article, we focus on this exciting and emerging role of F420 in catalyzing the oxidoreductive transformation of various imine, ketone and enoate moieties in secondary metabolites. Given the extensive and increasing availability of genomic and metagenomic data, these F420-dependent transformations may lead to the discovery of novel secondary metabolites, providing an invaluable and untapped resource in various biotechnological applications.</jats:p

Characterization of proline utilization pathway in Mycobacterium tuberculosis

The proline utilization pathway in Mycobacterium tuberculosis (Mtb) has been recently identified as an important factor in Mtb persistence in vivo, suggesting that this pathway could be a valuable therapeutic target against tuberculosis (TB). In Mtb, two distinct enzymes perform the conversion of proline into glutamate; the first step is the oxidation of proline into Δ1-pyrroline-5-carboxylic acid (P5C) by the flavoenzyme proline dehydrogenase (PruB) and the second reaction involves converting the tautomeric form of P5C (glutamate-γ-semialdehyde) into glutamate using the NAD+-dependent Δ1-pyrroline-5-carboxylic dehydrogenase (PruA). Here we describe three-dimensional structures of Mtb-PruA, determined by X-ray crystallography both in its apo state and in complex with NAD+ at 2.5 and 2.1 Å resolution, respectively. The structure reveals a conserved NAD+ binding mode, common to other related enzymes. Conformational differences in the active site, however, linked to changes in the dimer interface, suggest possibilities for selective inhibition of Mtb-PruA despite reasonably high sequence identity with other PruA enzymes. Using recombinant PruA and PruB, the proline utilization pathway in Mtb has also been reconstituted in vitro. Functional validation using a novel NMR approach has demonstrated that the PruA and PruB enzymes are together sufficient to convert proline to glutamate, the first such demonstration for monofunctional proline utilization enzymes.</jats:p