Ghader Bashiri

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Verified

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• PhD
• Professor (Associate) at University of Auckland

In the bacterium M. smegmatis , an enzyme called MftG allows the cofactor mycofactocin to transfer electrons released during ethanol metabolism to the electron transport chain.

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 fo...

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 pr...

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 stru...

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,...

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 epoxi...

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 t...

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,...

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 s...

Abstratct 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...

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 prot...

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.

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 discover...

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 bee...

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...

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. t...

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 ecol...

Mycobacterium tuberculosis isocitrate lyases (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.

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. t...

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‐t...

Lexapeptide is a lantibiotic showing potent antibacterial activity against Gram‐positive bacteria such as MRSA and MRSE, featured by the lanthionine (Lan) and d‐Ala formation in its structure. The formation of Lan is catalyzed by a class V lanthionine synthetase comprised of three standalone proteins. The d‐Ala28 is installed by the F420H2‐dependen...

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 unclea...

Lincosamide antibiotics, antitumor pyrrolobenzodiazepines (PBDs), quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these metabolites is the incorporation of L-tyrosine- or L-leucine-derived 4-alkyl-L-proline derivatives (APDs) i...

Menaquinone (Vitamin K2) plays a vital role in energy generation and environmental adaptation in many bacteria, including Mycobacterium 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...

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...

Iron-sulfur (Fe-S) 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 Fe-S clusters have non-redox roles in the active sites of enzymes. Such clusters are often prone to oxidative degradation, making the enzymes difficult to characterize....

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 h...

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 h...

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 info...

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 typ...

F 420 ‐dependent glucose‐6‐phosphate dehydrogenase (FGD) is a Mycobacterial enzyme that catalyzes the conversion of glucose‐6‐phosphate (G6P) to 6‐phosphogluconolactone using oxidized F 420 . This enzyme is important within Mycobacteria tuberculosis , which is the causative agent of tuberculosis disease. Our work has focused on probing the FGD reac...

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...

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 usi...

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. Isoc...

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...

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...

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 proceed...

Engineering cofactor specificity of enzymes is a promising approach that can expand the application of enzymes for biocatalytic production of industrially relevant chemicals. Until now, only NADPH-dependent imine reductases (IREDs) are known. This limits their applications to reactions employing whole cells as a cost-efficient cofactor regeneration...

Tuberculosis disease (TB) is a deadly infectious disease that is caused by Mycobacterium tuberculosis ( Mtb ). Our study focuses on F 420 ‐dependent glucose‐6‐phosphate dehydrogenase (FGD), the enzyme that catalyzes the first committed step of the pentose phosphate pathway within Mtb . FGD uses the oxidized F 420 cofactor to convert glucose‐6‐phosp...

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...

Pyridoxal 5'-phosphate (PLP) biosynthesis is essential for the survival and virulence of M. 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)-indepen...

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have bee...

Adiponectin, a collagenous hormone secreted abundantly from adipocytes, possesses potent anti-diabetic and anti-inflammatory properties. Mediated by the conserved Cys39 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-...

Significance Racemic protein crystallography was used to determine the X-ray structure of the predicted Mycobacterium tuberculosis protein Rv1738, which had been completely recalcitrant to crystallization in its natural l -form. Native chemical ligation was used to synthesize both l -protein and d -protein enantiomers of Rv1738. Crystallization of...

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. 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 shortco...

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 oxidat...

Phosphopantetheinyl transferases (PPTases) are essential enzymes that catalyze covalent attachment of the 4'-phosphopantetheine (4'-PP) moiety from coenzyme A (CoA) to a conserved serine residue on acyl (ACP) and peptidyl carrier proteins (PCP) [1]. This post-translational modification converts the inactive apo-carrier proteins to the functional fo...

The proline-utilization pathway in Mycobacterium tuberculosis (Mtb) has recently been 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 oxidat...

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 th...

F(420) is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F(420) 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 seque...

Primers used in the amplification of different Rv0132c constructs. (DOCX)

Supplementary methods. Details of bacterial growth; PCR amplification, cloning and preparation of constructs; western blotting; immunoelectron microscopy and homology modeling. (DOC)

Calibration curve for estimation of Rv0132c–Δ38 molecular weight using analytical SEC. The molecular weight calibration curve was obtained by plotting Kav values against LogMW of protein standards. The Kav value determined from the elution volume of Rv0132c–Δ38, indicated with *, corresponded to a molecular weight of 75.4 kDa, which is indicative o...

Mycobacterial strains and plasmids used in this study. (DOCX)

During cofactor F(420) biosynthesis, the enzyme F(420)-γ-glutamyl ligase (FbiB) catalyzes the addition of γ-linked L-glutamate residues to form polyglutamylated F(420) derivatives. In Mycobacterium tuberculosis, Rv3262 (FbiB) consists of two domains: an N-terminal domain from the F(420) ligase superfamily and a C-terminal domain with sequence simil...

Cofactor F(420) is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F(420) has a direct and important role in archaeal energy metabolism whereas the role of F(420) in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F(...

PA-824 is a 2-nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F(420) )-dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des-nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti-mycobacteri...

FGD1 is an F{sub 420}-dependent glucose-6-phosphate dehydrogenase from Mycobacterium tuberculosis that has been shown to be essential for activation of the anti-TB compound PA-824. Initial attempts to produce recombinant FGD1 using Escherichia coli as a host was unsuccessful, but when the alternative host Mycobacterium smegmatis was used, soluble p...

The modified flavin coenzyme F is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F-dependent glucose-6-phosphate dehydrogenase FG...

The modified flavin coenzyme F420 is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F420-dependent glucose-6-phosphate dehydrogen...

A major obstacle associated with recombinant protein over-expression in Escherichia coli is the production of insoluble inclusion bodies, a problem particularly pronounced with Mycobacterium tuberculosis proteins. One strategy to overcome the formation of inclusion bodies is to use an expression host that is more closely related to the organism fro...

FGD1 is an F(420)-dependent glucose-6-phosphate dehydrogenase from Mycobacterium tuberculosis that has been shown to be essential for activation of the anti-TB compound PA-824. Initial attempts to produce recombinant FGD1 using Escherichia coli as a host was unsuccessful, but when the alternative host Mycobacterium smegmatis was used, soluble prote...