Crystal Structures of F420-dependent Glucose-6-phosphate Dehydrogenase FGD1 Involved in the Activation of the Anti-tuberculosis Drug Candidate PA-824 Reveal the Basis of Coenzyme and Substrate Binding

Regimens targeting Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of M. tuberculosis demonstrates the need for additional TB-specific drugs. A notable feature of M. tuberculosis is coenzyme F 420 , which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on F 420 biosynthesis nominated many actinobacterial proteins as candidate F 420 -dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was determined to be limited to F 420 -producing species. The LLM and PPOX families were observed in F 420 -producing species as well as species lacking F 420 but were particularly numerous in many actinobacterial species, including M. tuberculosis. Partitioning the LLM and PPOX families based on an organism's ability to make F 420 allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify F 420 -correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that M. tuberculosis carries at least 28 separate F 420 -dependent enzymes, most of unknown function, and a paucity of flavin mononucleotide (FMN)-dependent proteins in these families. While prevalent in mycobacteria, markers of F 420 biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that M. tuberculosis relies heavily on coenzyme F 420 for its redox reactions. This dependence and the cofactor's rarity may make F 420 -related proteins promising drug targets.

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“…3B4Y [4]). Peak 1, SDH, is in contact with the carboxylate oxygens of the deazaflavin terminal ring (cyan).…”

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confidence: 99%

Unexpected Abundance of Coenzyme F ₄₂₀ -Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria

2010

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“…Both of these reactions use NAD ϩ or NADP ϩ , respectively, as cofactors rather than FAD as was shown for GilR. However, an F 420 -dependent glucose-6-phosphate dehydrogenase (FGD1 or FGD2) from Mycobacterium tuberculosis catalyzes a flavin-dependent hemiacetal to lactone conversion (44). As the closest related protein with the same function found in the literature, we compared the crystal structures of FGD1 and GilR but found no structural conservation.…”

Section: Resultsmentioning

confidence: 99%

The Crystal Structure and Mechanism of an Unusual Oxidoreductase, GilR, Involved in Gilvocarcin V Biosynthesis

Noinaj

Bosserman

Schickli

et al. 2011

Journal of Biological Chemistry

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GilR is a recently identified oxidoreductase that catalyzes the terminal step of gilvocarcin V biosynthesis and is a unique enzyme that establishes the lactone core of the polyketide-derived gilvocarcin chromophore. Gilvocarcin-type compounds form a small distinct family of anticancer agents that are involved in both photo-activated DNA-alkylation and histone H3 cross-linking. High resolution crystal structures of apoGilR and GilR in complex with its substrate pregilvocarcin V reveals that GilR belongs to the small group of a relatively new type of the vanillyl-alcohol oxidase flavoprotein family characterized by bicovalently tethered cofactors. GilR was found as a dimer, with the bicovalently attached FAD cofactor mediated through His-65 and Cys-125. Subsequent mutagenesis and functional assays indicate that Tyr-445 may be involved in reaction catalysis and in mediating the covalent attachment of FAD, whereas Tyr-448 serves as an essential residue initiating the catalysis by swinging away from the active site to accommodate binding of the 6R-configured substrate and consequently abstracting the proton of the hydroxyl residue of the substrate hemiacetal 6-OH group. These studies lay the groundwork for future enzyme engineering to broaden the substrate specificity of this bottleneck enzyme of the gilvocarcin biosynthetic pathway for the development of novel anti-cancer therapeutics.

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“…The addition of further L-glutamate residues to F 420 -2 is evidently the rate-limiting step of the process because it takes a much longer time for the F 420 species with longer poly-␥-glutamate tails to appear. When F 420 is purified from mycobacterial cells, the major species found contain between 5 and 7 L-glutamate residues in their poly-␥-glutamate tails (3,19). In addition, the full-length FbiB protein expressed in M. smegmatis shows co-purification of F 420 molecules with up to 11 L-glutamate residues incorporated, with the major species having 6 -9 residues.…”

Section: Discussionmentioning

confidence: 99%

“…F 420 has been emerging as a new player in the biology of mycobacteria (1), with increasing numbers of F 420 -utilizing proteins characterized from different mycobacterial species (2)(3)(4)(5)(6)(7)(8). This cofactor has been suggested to protect Mycobacterium tuberculosis, the causative agent of tuberculosis, against oxidative and nitrosative stress during pathogenesis (9 -11).…”

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confidence: 99%

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis

Bashiri

Rehan

Sreebhavan

et al. 2016

Journal of Biological Chemistry

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Cofactor F 420 is an electron carrier with a major role in the oxidoreductive reactions of Mycobacterium tuberculosis, the causative agent of tuberculosis. A ␥-glutamyl ligase catalyzes the final steps of the F 420 biosynthesis pathway by successive additions of L-glutamate residues to F 420 -0, producing a poly-␥-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F 420 -2 as the major species. The homologous M. tuberculosis enzyme, FbiB, is a two-domain protein and produces F 420 with predominantly 5-7 L-glutamate residues in the poly-␥-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated ␥-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 F 420 -0 in vitro to produce F 420 -5 after 24 h; communication between the two domains is critical for full ␥-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F 420 -0-, and FMN-bound states, displaying distinct sites for F 420 -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 and its implications for the role of N-and C-terminal domains in catalysis.The cofactor F 420 is a flavin derivative that is sporadically distributed among microorganisms, mainly archaea and actinobacteria (including mycobacteria). F 420 has been emerging as a new player in the biology of mycobacteria (1), with increasing numbers of F 420 -utilizing proteins characterized from different mycobacterial species (2-8). This cofactor has been suggested to protect Mycobacterium tuberculosis, the causative agent of tuberculosis, against oxidative and nitrosative stress during pathogenesis (9 -11). At the biochemical level, cofactor F 420 functions as a hydride transfer agent in oxidoreductive reactions with a lower redox potential than that of NAD(P) ϩ (12). The biosynthesis pathway of cofactor F 420 has been investigated in both archaeal and mycobacterial species. In the current view of the proposed pathway, the first intermediate with the complete chromophore (7, ) is produced by FO synthase (FbiC in mycobacteria (13) and CofGH in archaea (14)). A transferase enzyme (FbiA in mycobacteria (15) and CofD in archaea (16)) subsequently catalyzes the addition of a 2-phospho-L-lactate moiety to FO to produce F 420 -0 (F 420 with no poly-␥-glutamate tail). The final step of the pathway is performed by a ␥-glutamyl ligase (FbiB in mycobacteria (15) and CofE in archaea (17)) that catalyzes successive additions of L-glutamate residues to F 420 -0 (Fig. 1A).The length of the poly-␥-glutamate tail varies between archaeal and mycobacterial species; in archaea, two L-glutamate residues are seen (18), whereas in mycobacteria, up to nine residues are present (3, 19). There exists an intriguing difference between the ...

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Crystal Structures of F420-dependent Glucose-6-phosphate Dehydrogenase FGD1 Involved in the Activation of the Anti-tuberculosis Drug Candidate PA-824 Reveal the Basis of Coenzyme and Substrate Binding

Cited by 92 publications

References 48 publications

Unexpected Abundance of Coenzyme F ₄₂₀ -Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria

Unexpected Abundance of Coenzyme F ₄₂₀ -Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria

The Crystal Structure and Mechanism of an Unusual Oxidoreductase, GilR, Involved in Gilvocarcin V Biosynthesis

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis

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Crystal Structures of F420-dependent Glucose-6-phosphate Dehydrogenase FGD1 Involved in the Activation of the Anti-tuberculosis Drug Candidate PA-824 Reveal the Basis of Coenzyme and Substrate Binding

Cited by 92 publications

References 48 publications

Unexpected Abundance of Coenzyme F 420 -Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria

Unexpected Abundance of Coenzyme F 420 -Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria

The Crystal Structure and Mechanism of an Unusual Oxidoreductase, GilR, Involved in Gilvocarcin V Biosynthesis

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis

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Unexpected Abundance of Coenzyme F ₄₂₀ -Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria

Unexpected Abundance of Coenzyme F ₄₂₀ -Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria