Demonstration that fbiC is required by Mycobacterium bovis BCG for coenzyme F(420) and FO biosynthesis

Using the nitroimidazopyran-based antituberculosis drug PA-824 as a selective agent, transposon-generated Mycobacterium bovis strain BCG (M. bovis) mutants that could not make coenzyme F(420) were identified. Four independent mutants that could not make F(420) or the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in the M. bovis homologue of the Mycobacterium tuberculosis gene Rv1173, which we have named fbiC. Complementation of an M. bovis FbiC(-) mutant with fbiC restored the F(420) phenotype. These data demonstrate that fbiC is essential for F(420) production and that FbiC participates in a portion of the F(420) biosynthetic pathway between pyrimidinedione and FO. Homologues of fbiC were found in all 11 microorganisms that have been fully sequenced and that are known to make F(420). Four of these homologues (all from members of the aerobic actinomycetes) coded for proteins homologous over the entire length of the M. bovis FbiC, but in seven microorganisms two separate genes were found to code for proteins homologous with either the N-terminal or C-terminal portions of the M. bovis FbiC. Histidine-tagged FbiC overexpressed in Escherichia coli produced a fusion protein of the molecular mass predicted from the M. bovis BCG sequence (approximately 95,000 Da), as well as three other histidine-tagged proteins of significantly smaller size, which are thought to be proteolysis products of the FbiC fusion protein.     

Structure of Ddn, the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis involved in bioreductive activation of PA-824

Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.     

Large-scale production of coenzyme F420-5,6 by using Mycobacterium smegmatis

Production of coenzyme F420 and its biosynthetic precursor FO was examined with a variety of aerobic actinomycetes to identify an improved source for these materials. Based on fermentation costs, safety, and ease of growth, Mycobacterium smegmatis was the best source for F420-5,6. M. smegmatis produced 1 to 3 micromol of intracellular F420 per liter of culture, which was more than the 0.85 to 1.0 micromol of F420-2 per liter usually obtained with Methanobacterium thermoautotrophicum and approximately 10-fold higher than what was previously reported for the best aerobic actinomycetes. An improved chromatography system using rapidly flowing quaternary aminoethyl ion-exchange material and Florisil was used to more quickly and easily purify F420 than with previous methods.     

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

The modified flavin coenzyme F(420) 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(420)-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F(420) for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F(420) and citrate at resolutions of 1.90 and 1.95 A(,) respectively. The structure reveals a highly specific F(420) binding mode, which is shared with several other F(420)-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F(420) and is shown to be a competitive inhibitor (IC(50) 43 microm). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F(420) and helps promote a butterfly bend conformation that facilitates F(420) reduction and catalysis.     

Structures of coenzyme F(420) in Mycobacterium species

The structure of coenzyme F(420) in Mycobacterium smegmatis was examined using proton NMR, amino acid analysis, and HPLC. The two major F(420) structures were shown to be composed of a chromophore identical to that of F(420) from Methanobacterium thermoautotrophicum, with a side chain of a ribityl residue, a lactyl residue and five or six glutamate groups (F(420)-5 and F(420)-6). Peptidase treatment studies suggested that L-glutamate groups are linked by gamma-glutamyl bonds in the side chain. HPLC analysis indicated that Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium fortuitum have F(420)-5 and F(420)-6 as the predominant structures, whereas Mycobacterium avium contains F(420)-5, F(420)-6 and F(420)-7 in significant amounts. 7,8-Didemethyl 8-hydroxy 5-deazariboflavin (FO), an intermediate in F(420) biosynthesis, accounted for about 1-7% of the total deazaflavin in cells. Peptidase treatment of F(420) created F(420) derivatives that may be useful for the assay of enzymes involved in F(420) biosynthesis.     

Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.     

Expression, purification and crystallization of native and selenomethionine labeled Mycobacterium tuberculosis FGD1 (Rv0407) using a Mycobacterium smegmatis expression system

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 protein yields of 7 mg/L of culture were achieved. Both native and selenomethionine-substituted FGD1 were obtained by culturing M. smegmatis in autoinduction media protocols originally developed for E. coli. Using these media afforded the advantages of decreased handling, as cultures did not require monitoring of optical density and induction, and reduced cost by removing the need for expensive ADC enrichment normally used in mycobacterial cultures. Selenomethionine was efficiently incorporated at levels required for multiwavelength anomalous diffraction experiments used in crystal structure determination. As far as we are aware this is the first protocol for preparation of selenomethionine-substituted protein in mycobacteria. Native and selenomethionine-labeled FGD1 were successfully crystallized by vapor diffusion, with the crystals diffracting to 2.1 Angstrom resolution.     

Re-evaluation of the function of the F420 dehydrogenase in electron transport of Methanosarcina mazei

Methanosarcina mazei is a methanogenic archaeon that is able to thrive on various substrates and therefore contains a variety of redox-active proteins involved in both cytoplasmic and membrane-bound electron transport. The organism possesses a complex branched respiratory chain that has the ability to utilize different electron donors. In this study, two knockout mutants of the membrane-bound F(420) dehydrogenase (ΔfpoF and ΔfpoA-O) were constructed and analyzed. They exhibited severe growth deficiencies with trimethylamine, but not with acetate, as substrates. In cell lysates of the fpo mutants, the F(420):heterodisulfide oxidoreductase activity was strongly reduced, although soluble F(420) hydrogenase was still present. This led to the conclusion that the predominant part of cellular oxidation of the reduced form of F(420) (F(420)H(2)) in Ms. mazei is performed by F(420) dehydrogenase. Enzyme assays of cytoplasmic fractions revealed that ferredoxin (Fd):F(420) oxidoreductase activity was essentially absent in the ΔfpoF mutant. Subsequently, FpoF was produced in Escherichia coli and purified for further characterization. The purified FpoF protein catalyzed the Fd:F(420) oxidoreductase reaction with high specificity (the K(M) for reduced Fd was 0.5 μM) but with low velocity (V(max) = 225 mU·mg(-1)) and was present in the Ms. mazei cytoplasm in considerable amounts. Consequently, soluble FpoF might participate in electron carrier equilibrium and facilitate survival of the Ms. mazei Δech mutant that lacks the membrane-bound Fd-oxidizing Ech hydrogenase.     

Synthesis and antitubercular activity of 7-(R)- and 7-(S)-methyl-2-nitro-6-(S)-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazines, analogues of PA-824

Nitroimidazoles such as PA-824 and OPC-67683 are currently in clinical development as members of a promising new class of therapeutics for tuberculosis. While the antitubercular activity of these compounds is high, they both suffer from poor water solubility thus complicating development. We determined the single crystal X-ray structure of PA-824 and found a close packing of the nitroimidazoles facilitated by a pseudoaxial conformation of the p-trifluoromethoxybenzyl ether. To attempt to disrupt this tight packing by destabilizing the axial preference of this side chain, we prepared the two diastereomers of the 7-methyl-nitroimidazo-oxazine. Determination of the crystal structure of the 7-(S)-methyl derivative (5, cis) revealed that the benzylic side chain remained pseudoaxial while the 7-(R)-methyl derivative (6, trans) adopted the desired pseudoequatorial conformation. Both derivatives displayed similar activities against Mycobacterium tuberculosis, but neither showed improved aqueous solubility, suggesting that inherent lattice stability is not likely to be a major factor in limiting solubility. Conformational analysis revealed that all three compounds have similar energetically accessible conformations in solution. Additionally, these results suggest that the nitroreductase that initially recognizes PA-824 is somewhat insensitive to substitutions at the 7-position.     

Large-Scale Production of Coenzyme F420-5,6 by Using Mycobacterium smegmatis

Production of coenzyme F₄₂₀ and its biosynthetic precursor FO was examined with a variety of aerobic actinomycetes to identify an improved source for these materials. Based on fermentation costs, safety, and ease of growth, Mycobacterium smegmatis was the best source for F₄₂₀-5,6. M. smegmatis produced 1 to 3 μmol of intracellular F₄₂₀ per liter of culture, which was more than the 0.85 to 1.0 μmol of F₄₂₀-2 per liter usually obtained with Methanobacterium thermoautotrophicum and ~10-fold higher than what was previously reported for the best aerobic actinomycetes. An improved chromatography system using rapidly flowing quaternary aminoethyl ion-exchange material and Florisil was used to more quickly and easily purify F₄₂₀ than with previous methods.     

Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei.

Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant.     

Lack of mycothiol and ergothioneine induces different protective mechanisms in Mycobacterium smegmatis

Mycobacterium smegmatis contains the low molecular weight thiols, mycothiol (MSH) and ergothioneine (ESH). Examination of transposon mutants disrupted in mshC and egtA, involved in the biosynthesis of MSH and ESH respectively, demonstrated that both mutants were sensitive to oxidative, alkylating, and metal stress. However, the mshC mutant exhibited significantly more protein carbonylation and lipid peroxidation than wildtype, while the egtA mutant had less protein and lipid damage than wildtype. We further show that Ohr, KatN, and AhpC, involved in protection against oxidative stress, are upregulated in the egtA mutant. In the mshC mutant, an Usp and a putative thiol peroxidase are upregulated. In addition, mutants lacking MSH also contained higher levels of Coenzyme F420 as compared to wildtype and two Coenzyme F420 dependent enzymes were found to be upregulated. These results indicate that lack of MSH and ESH result in induction of different mechanisms for protecting against oxidative stress.     

The effect of 5-substitution on the electrochemical behavior and antitubercular activity of PA-824

Nitroimidazole PA-824 is part of an exciting new class of compounds currently undergoing clinical evaluation as novel TB therapeutics. The recently elucidated mechanism of action of PA-824 involves reduction of the nitroimidazole ring and subsequent nitric oxide release. The importance of this compound and its unique activity prompted us to explore how substitution of the nitroimidazole ring would affect electrochemical reduction and antitubercular activity. We prepared analogs of PA-824 with bromo, chloro, cyano, and amino substituents in the 5-position of the aromatic ring. We found that substitution of the imidazole ring greatly influences reduction and the stability of the corresponding nitro radical anion. Further, the antitubercular activities of the bromo and chloro analogs may indicate that an alternate nitroreductase pathway within Mycobacterium tuberculosis exists.     

Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei

Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant.     

Structural insights of PA-824 derivatives: ligand-based 3D-QSAR study and design of novel PA824 derivatives as anti-tubercular agents

Abstract

With the purpose of designing novel chemical entities with improved inhibitory potencies against drug-resistant Mycobacterium tuberculosis, the 3D- quantitative structure–activity relationship (QSAR) studies were carried out on biphenyl analogs of the tuberculosis (TB) drug, PA-824. Anti-mycobacterial activity (MABA) was considered for the 3D-QSAR studies using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) approaches. The best CoMFA and CoMSIA models were found statistically significant with cross-validated coefficients (q²) of 0.784 and 0.768, respectively, and conventional coefficients (r²) of 0.823 and 0.981, respectively. The cross-validated and the external validation results revealed that both the CoMFA and CoMSIA models possesses high accommodating capacities and they would be reliable for predicting the pMIC values of new PA-824 derivatives. Based on the models and structural insights, a series of new PA-824 derivatives were designed and the anti-mycobacterial activities of the designed compounds were predicted based on the best 3D-QSAR model. The predicted data results suggest the designed compounds are more potent than existed ones.     

Methanococcus jannaschii coenzyme F420 analogs contain a terminal alpha-linked glutamate

Analyses of the F(420)s present in Methanococcus jannaschii have shown that these cells contain a series of gamma-glutamyl-linked F(420)s capped with a single, terminal alpha-linked L-glutamate. The predominant form of F(420) was designated as alpha-F(420)-3 and represented 86% of the F(420)s in these cells. Analyses of Methanosarcina thermophila, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, and Mycobacterium smegmatis showed that they contained only gamma-glutamyl-linked F(420)s.     

In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis

In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Re)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.     

Mycothiol import by Mycobacterium smegmatis and function as a resource for metabolic precursors and energy production

Mycothiol ([MSH] AcCys-GlcN-Ins, where Ac is acetyl) is the major thiol produced by Mycobacterium smegmatis and other actinomycetes. Mutants deficient in MshA (strain 49) or MshC (transposon mutant Tn1) of MSH biosynthesis produce no MSH. However, when stationary phase cultures of these mutants were incubated in medium containing MSH, they actively transported it to generate cellular levels of MSH comparable to or greater than the normal content of the wild-type strain. When these MSH-loaded mutants were transferred to MSH-free preconditioned medium, the cellular MSH was catabolized to generate GlcN-Ins and AcCys. The latter was rapidly converted to Cys by a high deacetylase activity assayed in extracts. The Cys could be converted to pyruvate by a cysteine desulfhydrase or used to regenerate MSH in cells with active MshC. Using MSH labeled with [U-(14)C]cysteine or with [6-(3)H]GlcN, it was shown that these residues are catabolized to generate radiolabeled products that are ultimately lost from the cell, indicating extensive catabolism via the glycolytic and Krebs cycle pathways. These findings, coupled with the fact the myo-inositol can serve as a sole carbon source for growth of M. smegmatis, indicate that MSH functions not only as a protective cofactor but also as a reservoir of readily available biosynthetic precursors and energy-generating metabolites potentially important under stress conditions. The half-life of MSH was determined in stationary phase cells to be approximately 50 h in strains with active MshC and 16 +/- 3 h in the MshC-deficient mutant, suggesting that MSH biosynthesis may be a suitable target for drugs to treat dormant tuberculosis.     

Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> polyhydroxyalkanoate synthase

The F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaC_Re). We have now carried out site-directed saturation mutagenesis of F420 of PhaC_Re and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaC_Re enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaC_Re enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant.