F420-dependent transformations in biosynthesis of secondary metabolites.

Cofactor F420 has been historically known as the "methanogenic redox cofactor". It is now recognised that F420 has essential roles in the primary and secondary metabolism of archaea and bacteria. Recent discoveries highlight the role of F420 as a redox cofactor in the biosynthesis of various natural products, including ribosomally synthesised and post-translationally modified peptides, and a new class of nicotinamide adenine dinucleotide-based secondary metabolites. With the vast availability of (meta)genomic data, the identification of uncharacterised F420-dependent enzymes offers the potential for discovering novel secondary metabolites, presenting valuable prospects for clinical and biotechnological applications.

Poly-γ-glutamylation of biomolecules.

Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F420. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.

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

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.

Mycobacterium tuberculosis Rv1916 is an Acetyl-CoA-Binding Protein.

Isocitrate lyase (ICL) isoform 2 is an essential enzyme for some clinical Mycobacterium tuberculosis (Mtb) strains during infection. In the laboratory Mtb strain H37Rv, the icl2 gene encodes two distinct gene products - Rv1915 and Rv1916 - due to a frameshift mutation. This study aims to characterise these two gene products to understand their structure and function. While we were unable to produce Rv1915 recombinantly, soluble Rv1916 was obtained with sufficient yield for characterisation. Kinetic studies using UV-visible spectrophotometry and 1 H-NMR spectroscopy showed that recombinant Rv1916 does not possess isocitrate lyase activity, while waterLOGSY binding experiments demonstrated that it could bind acetyl-CoA. Finally, X-ray crystallography revealed structural similarities between Rv1916 and the C-terminal domain of ICL2. Considering the probable differences between full-length ICL2 and the gene products Rv1915 and Rv1916, care must be taken when using Mtb H37Rv as a model organism to study central carbon metabolism.

Allosteric inhibition of Staphylococcus aureus MenD by 1,4-dihydroxy naphthoic acid: a feedback inhibition mechanism of the menaquinone biosynthesis pathway.

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'.

Flavin-enabled reductive and oxidative epoxide ring opening reactions.

Epoxide ring opening reactions are common and important in both biological processes and synthetic applications and can be catalyzed in a non-redox manner by epoxide hydrolases or reductively by oxidoreductases. Here we report that fluostatins (FSTs), a family of atypical angucyclines with a benzofluorene core, can undergo nonenzyme-catalyzed epoxide ring opening reactions in the presence of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The 2,3-epoxide ring in FST C is shown to open reductively via a putative enol intermediate, or oxidatively via a peroxylated intermediate with molecular oxygen as the oxidant. These reactions lead to multiple products with different redox states that possess a single hydroxyl group at C-2, a 2,3-vicinal diol, a contracted five-membered A-ring, or an expanded seven-membered A-ring. Similar reactions also take place in both natural products and other organic compounds harboring an epoxide adjacent to a carbonyl group that is conjugated to an aromatic moiety. Our findings extend the repertoire of known flavin chemistry that may provide new and useful tools for organic synthesis.

AvmM catalyses macrocyclization through dehydration/Michael-type addition in alchivemycin A biosynthesis.

Macrocyclization is an important process that affords morphed scaffold in biosynthesis of bioactive natural products. Nature has adapted diverse biosynthetic strategies to form macrocycles. In this work, we report the identification and characterization of a small enzyme AvmM that can catalyze the construction of a 16-membered macrocyclic ring in the biosynthesis of alchivemycin A (1). We show through in vivo gene deletion, in vitro biochemical assay and isotope labelling experiments that AvmM catalyzes tandem dehydration and Michael-type addition to generate the core scaffold of 1. Mechanistic studies by crystallography, DFT calculations and MD simulations of AvmM reveal that the reactions are achieved with assistance from the special tenuazonic acid like moiety of substrate. Our results thus uncover an uncharacterized macrocyclization strategy in natural product biosynthesis.

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.

Clusterin is involved in mediating the metabolic function of adipose SIRT1.

SIRT1 is a metabolic sensor regulating energy homeostasis. The present study revealed that mice with selective overexpression of human SIRT1 in adipose tissue (Adipo-SIRT1) were protected from high-fat diet (HFD)-induced metabolic abnormalities. Adipose SIRT1 was enriched at mitochondria-ER contacts (MERCs) to trigger mitohormesis and unfolded protein response (UPRmt), in turn preventing ER stress. As a downstream target of UPRmt, clusterin was significantly upregulated and acted together with SIRT1 to regulate the protein and lipid compositions at MERCs of adipose tissue. In mice lacking clusterin, HFD-induced metabolic abnormalities were significantly enhanced and could not be prevented by overexpression of SIRT1 in adipose tissue. Treatment with ER stress inhibitors restored adipose SIRT1-mediated beneficial effects on systemic energy metabolism. In summary, adipose SIRT1 facilitated the dynamic interactions and communications between mitochondria and ER, via MERCs, in turn triggering a mild mitochondrial stress to instigate the defense responses against dietary obesity-induced metabolic dysfunctions.

ß-NAD as a building block in natural product biosynthesis.

ABSTRATCT: ß-Nicotinamide adenine dinucleotide (ß-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, ß-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 ß-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 ß-NAD and S-adenosylmethionine, generating a 6-azatetrahydroindane scaffold. Members of this novel family of ß-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 ß-NAD-derived natural products.

Lipid transport across the mycobacterial cell envelope.

The mammalian-cell-entry (Mce) proteins of Mycobacterium tuberculosis enable the bacterium to acquire lipids from the host cells. Asthana et al. [IUCrJ (2021). 8, 757-774] present the first structural insights into the potential assembly of Mce1 and Mce4, advancing our understanding of lipid transport by the human pathogen that causes tuberculosis.

Discovery and biosynthesis of guanipiperazine from a NRPS-like pathway.

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.

Itaconate is a covalent inhibitor of the Mycobacterium tuberculosis isocitrate lyase.

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.

Redox Modifications in the Biosynthesis of Alchivemycin A Enable the Formation of Its Key Pharmacophore.

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 ß-subunit of RNA polymerase. As further evidence, we show that AvmK, a ß-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.

Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis.

The nitroimidazole prodrugs 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, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, 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 (fbiD), 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.

Functional Genome Mining Reveals a Class V Lanthipeptide Containing a d-Amino Acid Introduced by an F420 H2 -Dependent Reductase.

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.

Convergent pathways to biosynthesis of the versatile cofactor F420.

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.

Allosteric regulation of menaquinone (vitamin K2) biosynthesis in the human pathogen Mycobacterium tuberculosis.

Menaquinone (vitamin K2) 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.

Different Reaction Specificities of F420H2-Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets.

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.