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Menaquinones (MKs) are electron carriers in bacterial respiratory chains. In Staphylococcus aureus ( Sau ), MKs are essential for aerobic and anaerobic respiration. As MKs are redox-active, their biosynthesis likely requires tight regulation to prevent disruption of cellular redox balance. We recently found that the Mycobacterium tuberculosis MenD, the first committed enzyme of the MK biosynthesis pathway, is allosterically inhibited by the downstream metabolite 1,4-dihydroxy-2-naphthoic acid (DHNA). To understand if this is a conserved mechanism in phylogenetically distant genera that also use MK, we investigated whether the Sau- MenD is allosterically inhibited by DHNA. Our results show that DHNA binds to and inhibits the SEPHCHC synthase activity of Sau -MenD enzymes. We identified residues in the DHNA binding pocket that are important for catalysis (Arg98, Lys283, Lys309) and inhibition (Arg98, Lys283). Furthermore, we showed that exogenous DHNA inhibits the growth of Sau , an effect that can be rescued by supplementing the growth medium with MK-4. Our results demonstrate that, despite a lack of strict conservation of the DHNA binding pocket between Mtb -MenD and Sau -MenD, feedback inhibition by DHNA is a conserved mechanism in Sau -MenD and hence the Sau MK biosynthesis pathway. These findings may have implications for the development of anti-staphylococcal agents targeting MK biosynthesis. This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.

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.

Itaconate is a mammalian antimicrobial metabolite that inhibits the isocitrate lyases (ICLs) of Mycobacterium tuberculosis. Herein, we report that ICLs form a covalent adduct with itaconate through their catalytic cysteine residue. These results reveal atomic details of itaconate inhibition and provide insights into the catalytic mechanism of ICLs.

Nonribosomal peptide synthetases (NRPSs) are modular enzymes that use a thiotemplate mechanism to assemble the peptide backbones of structurally diverse and biologically active natural products in bacteria and fungi. Unlike these canonical multi-modular NRPSs, single-module NRPS-like enzymes, which lack the key condensation (C) domain, are rare in bacteria, and have been largely unexplored to date. Here, we report the discovery of a gene cluster (gup) encoding a NRPS-like megasynthetase through genome mining. Heterologous expression of the gup cluster led to the production of two unprecedented alkaloids, guanipiperazines A and B. The NRPS-like enzyme activates two l-tyrosine molecules, reduces them to the corresponding amino aldehydes, and forms an unstable imine product. The subsequent enzymatic reduction affords piperazine, which can be morphed by a P450 monooxygenase into a highly strained compound through C–O bond formation. Further intermolecular oxidative coupling forming the C–C or C–O bond is catalyzed by another P450 enzyme. This work reveals the huge potential of NRPS-like biosynthetic gene clusters in the discovery of novel natural products., Genome mining of a NRPS-like gene cluster led to the identification of two novel alkaloids with antimicrobial activity. This work reveals the huge potential of NRPS-like biosynthetic gene clusters in the discovery of novel natural products.

Cofactor F420 is historically known as the methanogenic redox cofactor, having a key role in the central metabolism of methanogens, and archaea in general. Over the past decade, however, it has become evident this cofactor is more widely distributed across archaeal and bacterial taxa, suggesting a broader role for F420 in various metabolic and ecological capacities. In this article, we focus on the recent findings that have led to a deeper understanding of F420 biosynthetic enzymes and metabolites across microorganisms.

Lantibiotics are a type of ribosomally synthesized and post-translationally modified peptides (termed lanthipeptides) with often potent antimicrobial activity. Herein, we report the discovery of a new lantibiotic, lexapeptide, using the library expression analysis system (LEXAS) approach. Lexapeptide has rare structural modifications, including N-terminal (N,N)-dimethyl phenylalanine, C-terminal (2-aminovinyl)-3-methyl-cysteine, and d-Ala. The characteristic lanthionine moiety in lexapeptide is formed by three proteins (LxmK, LxmX, and LxmY), which are distinct from enzymes known to be involved in lanthipeptide biosynthesis. Furthermore, a novel F420 H2 -dependent reductase (LxmJ) from the lexapeptide biosynthetic gene cluster (BGC) is identified to catalyze the reduction of dehydroalanine to install d-Ala. Our findings suggest that lexapeptide is the founding member of a new class of lanthipeptides that we designate as class V. We also identified further class V lanthipeptide BGCs in actinomycetes and cyanobacteria genomes, implying that other class V lantibiotics await discovery.

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.

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.

β-Nicotinamide adenine dinucleotide (β-NAD) is a pivotal metabolite for all living organisms and functions as a diffusible electron acceptor and carrier in the catabolic arms of metabolism1,2. Furthermore, β-NAD is involved in diverse epigenetic, immunological and stress-associated processes, where it is known to be sacrificially utilized as an ADP-ribosyl donor for protein and DNA modifications, or the generation of cell-signalling molecules3,4. Here we report the function of β-NAD in secondary metabolite biosynthetic pathways, in which the nicotinamide dinucleotide framework is heavily decorated and serves as a building block for the assembly of a novel class of natural products. The gatekeeping enzyme of the discovered pathway (SbzP) catalyses a pyridoxal phosphate-dependent [3+2]-annulation reaction between β-NAD and S-adenosylmethionine, generating a 6-azatetrahydroindane scaffold. Members of this novel family of β-NAD-tailoring enzymes are widely distributed in the bacterial kingdom and are encoded in diverse biosynthetic gene clusters. The findings of this work set the stage for the discovery and exploitation of β-NAD-derived natural products. published

Redox enzymes play a critical role in transforming nascent scaffolds into structurally complex and biologically active natural products. Alchivemycin A (AVM, 1) is a highly oxidized polycyclic compound with potent antimicrobial activity and features a rare 2H-tetrahydro-4,6-dioxo-1,2-oxazine (TDO) ring system. The scaffold of AVM has previously been shown to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway. In this study, we present a postassembly secondary metabolic network involving six redox enzymes that leads to AVM formation. We characterize this complex redox network using in vivo gene deletions, in vitro biochemical assays, and one-pot enzymatic total synthesis. Importantly, we show that an FAD-dependent monooxygenase catalyzes oxygen insertion into an amide bond to form the key TDO ring in AVM, an unprecedented function of flavoenzymes. We also show that the TDO ring is essential to the antimicrobial activity of AVM, likely through targeting the β-subunit of RNA polymerase. As further evidence, we show that AvmK, a β-subunit of RNA synthase, can confer self-resistance to AVM via target modification. Our findings expand the repertoire of functions of flavoenzymes and provide insight into antimicrobial and biocatalyst development based on AVM.

The nitroimidazole pro-drugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: fbiC (56%), fbiA (15%), ddn (12%), fgd (4%) and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983, a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance, but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

Lantibiotics are a type of ribosomally synthesized and post-translationally modified peptides (termed lanthipeptides) with often potent antimicrobial activity. Herein, we report the discovery of a new lantibiotic, lexapeptide, using the library expression analysis system (LEXAS) approach. Lexapeptide has rare structural modifications, including N-terminal (N,N)-dimethyl phenylalanine, C-terminal (2-aminovinyl)-3-methyl-cysteine, and d-Ala. The characteristic lanthionine moiety in lexapeptide is formed by three proteins (LxmK, LxmX, and LxmY), which are distinct from enzymes known to be involved in lanthipeptide biosynthesis. Furthermore, a novel F

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F

Menaquinone (vitamin K(2)) plays a vital role in energy generation and environmental adaptation in many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb). Although menaquinone levels are known to be tightly linked to the cellular 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 that the last cytosolic metabolite in the menaquinone biosynthesis pathway, 1,4-dihydroxy-2-naphthoic acid (DHNA), binds to domain II of Mtb-MenD and inhibits its activity. Using X-ray crystallography of four apo- and cofactor-bound Mtb-MenD structures, along with several spectroscopy assays, we identified three arginine residues (Arg-97, Arg-277, and Arg-303) that are important for both enzyme activity and the feedback inhibition by DHNA. Among these residues, Arg-277 appeared 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, identifying a protein-level regulatory mechanism that controls menaquinone levels within the cell and may therefore represent a good target for disrupting menaquinone biosynthesis in M. tuberculosis.

Thiopeptins are highly decorated thiopeptide antibiotics similar in structure to thiostrepton A and harbor two unusual features. All thiopeptins contain a thioamide, a rare moiety among natural products, and a subset of thiopeptins present with a piperidine in the core macrocycle rather than the more oxidated dehydropiperidine or pyridine rings typically observed in the thiopeptides. Here, we report the identification of the thiopeptin biosynthetic gene (tpn) cluster in Streptomyces tateyamensis and the gene product, TpnL, which shows sequence similarity to (deaza)flavin-dependent oxidoreductases. Heterologous expression of TpnL in the thiostrepton A producer Streptomyces laurentii led to the production of a piperidine-containing analogue. Binding studies revealed that TpnL preferentially binds the deazaflavin cofactor coenzyme F(420), and in vitro reconstitution of TpnL activity confirmed that this enzyme is an F(420)H(2)-dependent dehydropiperidine reductase. The identification of TpnL and its activity establishes the basis for the piperidine-containing series a thiopeptides, one of the five main structural groups of this diverse family of antibiotics.

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.

Isocitrate lyase is important for lipid utilisation by Mycobacterium tuberculosis but its ICL2 isoform is poorly understood. Here we report that binding of the lipid metabolites acetyl-CoA or propionyl-CoA to ICL2 induces a striking structural rearrangement, substantially increasing isocitrate lyase and methylisocitrate lyase activities. Thus, ICL2 plays a pivotal role regulating carbon flux between the tricarboxylic acid (TCA) cycle, glyoxylate shunt and methylcitrate cycle at high lipid concentrations, a mechanism essential for bacterial growth and virulence., Isocitrate lyase (ICL) isoforms 1 and 2 are enzymes in the glyoxylate and methylcitrate cycles that enable Mycobacterium tuberculosis (Mtb) to use lipids as a carbon source. Here the authors present the ligand-free Mtb ICL2 and acetyl-CoA bound ICL2 crystal structures, which reveal a structural reorganisation upon acetyl-CoA binding that leads to an activation of its isocitrate lyase and methylcitrate lyase activities.

F420-dependent glucose-6-phosphate dehydrogenase (FGD) is involved in the committed step of the pentose phosphate pathway within mycobacteria, where it catalyzes the reaction between glucose-6-phosphate (G6P) and the F420 cofactor to yield 6-phosphogluconolactone and the reduced cofactor, F420H2. Here, we aim to probe the FGD reaction mechanism using dead-end inhibition experiments, as well as solvent and substrate deuterium isotope effects studies. The dead-end inhibition studies performed using citrate as the inhibitor revealed competitive and uncompetitive inhibition patterns for G6P and F420 respectively, thus suggesting a mechanism of ordered addition of substrates in which the F420 cofactor must first bind to FGD before G6P binding. The solvent deuterium isotope effects studies yielded normal solvent kinetic isotope effects (SKIE) on kcat and kcat/Km for both G6P and F420. The proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed SKIE is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P-d1 yielded KIE of 1.1 for both kcat and kcat/Km, while the pre-steady state KIE on kobs was 1.4. Because the hydride transferred to C5 of F420 was the one targeted for isotopic substitution, these KIE values provide further evidence to support our previous findings that hydride transfer is likely not rate-limiting in the FGD reaction [1].

F420-dependent glucose-6-phosphate dehydrogenase (FGD1) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone. During the course of this reaction, the oxidized cofactor F420 is converted to its reduced form (F420H2). FGD was initially identified and studied extensively within mycobacteria prior to the more recent discovery of FGDs from Rhodococcus jostii RHA1. Another group of FGDs that utilize a variety of sugar-6-phosphates from Nocardia and Cryptosporangium arvum is also known as the FSDs. Crystal structures of FGD from the pathogens Mycobacterium tuberculosis and Rhodococcus jostii RHA1 have provided significant insight into the structural and mechanistic properties of the enzymes and have been the basis for mutagenesis, kinetic and mechanistic studies in recent years. This review discusses the role of FGD within M. tuberculosis, followed by our current understanding of the FGD catalytic mechanism. This will also be discussed with reference to the relationship between FGD and the structurally unrelated NADP+-dependent glucose-6-phosphate enzymes.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that can remain dormant for many years before becoming active. One way to control and eliminate TB is the identification and treatment of latent TB, preventing infected individuals from developing active TB and thus eliminating the subsequent spread of the disease. Isocitrate lyase (ICL) is involved in the mycobacterial glyoxylate and methylisocitrate cycles. ICL is important for the growth and survival of M. tuberculosis during latent infection. ICL is not present in humans and is therefore a potential therapeutic target for the development of anti-TB agents. Here, we explore the evidence linking ICL to persistent survival of M. tuberculosis. The structure, mechanism and inhibition of the enzyme is also discussed.

Phosphopantetheinyl transferases (PPTases) are key elements in the modular syntheses performed by multienzyme systems such as polyketide synthases. PPTases transfer phosphopantetheine derivatives from Coenzyme A to carrier proteins (CPs), thus orchestrating substrate supply. We describe an efficient mass spectrometry‐based protocol for determining CP specificity for a particular PPTase in organisms possessing several candidate PPTases. We show that the CPs MbtL and PpsC, both involved in synthesis of essential metabolites in Mycobacterium tuberculosis, are exclusively activated by the type 2 PPTase PptT and not the type 1 AcpS. The assay also enables conclusive identification of the reactive serine on each CP.

Iron–sulfur clusters are protein cofactors with an ancient evolutionary origin. These clusters are best known for their roles in redox proteins such as ferredoxins, but some iron–sulfur clusters have nonredox roles in the active sites of enzymes. Such clusters are often prone to oxidative degradation, making the enzymes difficult to characterize. Here we report a structural and functional characterization of dihydroxyacid dehydratase (DHAD) from Mycobacterium tuberculosis (Mtb), an essential enzyme in the biosynthesis of branched-chain amino acids. Conducting this analysis under fully anaerobic conditions, we solved the DHAD crystal structure, at 1.88 Å resolution, revealing a 2Fe–2S cluster in which one iron ligand is a potentially exchangeable water molecule or hydroxide. UV and EPR spectroscopy both suggested that the substrate binds directly to the cluster or very close to it. Kinetic analysis implicated two ionizable groups in the catalytic mechanism, which we postulate to be Ser-491 and the iron-bound water/hydroxide. Site-directed mutagenesis showed that Ser-491 is essential for activity, and substrate docking indicated that this residue is perfectly placed for proton abstraction. We found that a bound Mg(2+) ion 6.5 Å from the 2Fe–2S cluster plays a key role in substrate binding. We also identified a putative entry channel that enables access to the cluster and show that Mtb-DHAD is inhibited by a recently discovered herbicide, aspterric acid, that, given the essentiality of DHAD for Mtb survival, is a potential lead compound for the design of novel anti-TB drugs.

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., Cofactor F420 plays crucial roles in bacterial and archaeal metabolism, but its biosynthetic pathway is not fully understood. Here, the authors present the structure of one of the enzymes and provide experimental evidence for a substantial revision of the pathway, including the identification of a new intermediate.

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.

Delamanid represents one of two novel antimicrobial classes approved to treat tuberculosis in over 40 years. Pretomanid is another promising nitroimidazole pro-drug in clinical development. Characterization of the full spectrum of mutations conferring resistance to nitroimidazoles and their related phenotypes in Mycobacterium tuberculosis will inform development of suitable genotypic and phenotypic drug susceptibility tests. Here, we used a range of pretomanid doses to select pretomanid-resistant mutants in two pathologically distinct murine TB models. The frequency of spontaneous pretomanid resistance mutations was approximately 10−5 CFU. Pretomanid demonstrated dose-dependent bactericidal activity and selective amplification of resistant mutants. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 90% of which were found in 1 of 5 genes previously associated with nitroimidazole activation and resistance. The remaining 10% harbored isolated mutations in Rv2983. Complementing an Rv2983 mutant with a wild-type copy of Rv2983 restored wild-type susceptibility to pretomanid and delamanid, confirming that loss of Rv2983 function causes nitroimidazole resistance. By quantifying F420 and its precursor Fo in Mycobacterium smegmatis overexpressing Rv2983 and an M. tuberculosis Rv2983 mutant, we provide evidence that Rv2983 is necessary for F420 biosynthesis and nitroimidazole activation, perhaps as the guanylyltransferase CofC. F420H2-deficient mutants displayed hypersusceptibility to malachite green (MG), a selective decontaminant present in solid media used to isolate and propagate mycobacteria from clinical samples. The wide diversity of mutations causing high-level pretomanid resistance and MG hypersusceptibility of most mutants poses significant challenges to clinical detection of nitroimidazole resistance using either genotypic or phenotypic methods.SignificanceNitroimidazole pro-drugs represent a promising new class of anti-tuberculosis drugs. Reliable methods to assure nitroimidazole susceptibility are critical to assure their optimal use. Yet, the spectrum of nitroimidazole resistance mutations remains incompletely characterized. Using 161 pretomanid-resistant Mycobacterium tuberculosis isolates selected in pretomanid-treated mice, we discovered a novel resistance determinant, Rv2983, required for cofactor F420 biosynthesis and characterized the remarkable diversity of mutations in this and 5 other genes involved in nitroimidazole activation. We show that F420H2–deficient nitroimidazole-resistant mutants are hypersusceptible to the selective decontaminant malachite green used in solid media to isolate mycobacteria and may evade detection on such media. These results have important implications for development and clinical use of genotypic and phenotypic methods for nitroimidazole susceptibility testing.

Menaquinone (MQ) is an essential component of the respiratory chains of many pathogenic organisms, including Mycobacterium tuberculosis (Mtb). The first committed step in MQ biosynthesis is catalyzed by 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD), a thiamin diphosphate (ThDP)-dependent enzyme. Catalysis proceeds through two covalent intermediates as the substrates 2-oxoglutarate and isochorismate are successively added to the cofactor before final cleavage of the product. We have determined a series of crystal structures of Mtb-MenD that map the binding of both substrates, visualizing each step in the MenD catalytic cycle, including both intermediates. ThDP binding induces a marked asymmetry between the coupled active sites of each dimer, and possible mechanisms of communication can be identified. The crystal structures also reveal conformational features of the two intermediates that facilitate reaction but prevent premature product release. These data fully map chemical space to inform early-stage drug discovery targeting MenD.

Pyridoxal 5'-phosphate (PLP) biosynthesis is essential for the survival and virulence of Mycobacterium tuberculosis (Mtb). PLP functions as a cofactor for 58 putative PLP-binding proteins encoded by the Mtb genome and could also act as a potential antioxidant. De novo biosynthesis of PLP in Mtb takes place through the 'deoxyxylulose 5'-phosphate (DXP)-independent' pathway, whereas PdxH enzymes, possessing pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPOx) activity, are involved in the PLP salvage pathway. In this study, we demonstrate that the annotated PdxH enzymes from various mycobacterial species are bona fide members of the classical PNPOx enzyme family, capable of producing PLP using both pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) substrates.

Adiponectin, a collagenous hormone secreted abundantly from adipocytes, possesses potent antidiabetic and anti-inflammatory properties. Mediated by the conserved Cys(39) located in the variable region of the N terminus, the trimeric (low molecular weight (LMW)) adiponectin subunit assembles into different higher order complexes, e.g. hexamers (middle molecular weight (MMW)) and 12-18-mers (high molecular weight (HMW)), the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. The endoplasmic reticulum (ER) chaperone ERp44 retains adiponectin in the early secretory compartment and tightly controls the oxidative state of Cys(39) and the oligomerization of adiponectin. Using cellular and in vitro assays, we show that ERp44 specifically recognizes the LMW and MMW forms but not the HMW form. Our binding assays with short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW form, into reduced trimeric precursors. These ERp44-bound precursors in the cis-Golgi may be transported back to the ER and released to enhance the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control.

The use of small molecules as “silver bullets” that can bind to generate crosslinks between protein molecules has been advanced as a powerful means of enhancing success in protein crystallization (McPherson and Cudney, 2006). We have explored this approach in attempts to overcome an order–disorder phenomenon that complicated the structural analysis of the enzyme Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis (P5CDH, Mtb-PruA). Using the Silver Bullets Bio screen, we obtained new crystal packing using cobalamin as a co-crystallization agent. This crystal form did not display the order–disorder phenomenon previously encountered. Solution of the crystal structure showed that cobalamin molecules are present in the crystal contacts. Although the cobalamin binding probably does not have physiological relevance, it reflects similarities in the nucleotide-binding region of Mtb-PruA, with the nucleotide loop of cobalamin sharing the binding site for the adenine moiety of NAD+.

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.

The enzymes isocitrate lyase (ICL) isoforms 1 and 2 are essential for Mycobacterium tuberculosis survival within macrophages during latent tuberculosis (TB). As such, ICLs are attractive therapeutic targets for the treatment of tuberculosis. However, there are few biophysical assays that are available for accurate kinetic and inhibition studies of ICL in vitro. Herein we report the development of a combined NMR spectroscopy and thermal shift assay to study ICL inhibitors for both screening and inhibition constant (IC50) measurement. Operating this new assay in tandem with virtual high-throughput screening has led to the discovery of several new ICL1 inhibitors.

The proline-utilization pathway inMycobacterium tuberculosis(Mtb) has recently been identified as an important factor inMtbpersistencein vivo, suggesting that this pathway could be a valuable therapeutic target against tuberculosis (TB). InMtb, 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, the three-dimensional structures ofMtb-PruA, determined by X-ray crystallography, in the apo state and in complex with NAD+are described at 2.5 and 2.1 Å resolution, respectively. The structure reveals a conserved NAD+-binding mode, common to other related enzymes. Species-specific conformational differences in the active site, however, linked to changes in the dimer interface, suggest possibilities for selective inhibition ofMtb-PruA despite its reasonably high sequence identity to other PruA enzymes. Using recombinant PruA and PruB, the proline-utilization pathway inMtbhas also been reconstitutedin 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.

Tetracyclines are a group of natural products sharing a linearly fused four-ring scaffold, which is essential for their broad-spectrum antibiotic activities. Formation of the key precursor anhydrotetracycline 3 during oxytetracycline 1 biosynthesis has been previously characterized. However, the enzymatic steps that transform 3 into 1, including the additional hydroxylation at C5 and the final C5a-C11a reduction, have remained elusive. Here we report two redox enzymes, OxyS and OxyR, are sufficient to convert 3 to 1. OxyS catalyzes two sequential hydroxylations at C6 and C5 positions of 3 with opposite stereochemistry, while OxyR catalyzes the C5a-C11a reduction using F420 as a cofactor to produce 1. The crystal structure of OxyS was obtained to provide insights into the tandem C6- and C5-hydroxylation steps. The substrate specificities of OxyS and OxyR were shown to influence the relative ratio of 1 and tetracycline 2.