Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0026918 (Mycobacterium)
 52,428 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The front-line antituberculosis drug isoniazid (INH) and the related drug ethionamide (ETH) are prodrugs that upon activation inhibit the synthesis of mycolic acids, leading to bactericidal activity. Coresistance to INH and ETH can be mediated by dominant mutations in the target gene inhA, encoding an enoyl-ACP reductase, or by recessive mutations in ndh, encoding a type II NADH dehydrogenase (NdhII). To address the mechanism of resistance mediated by the latter, we have isolated novel ndh mutants of Mycobacterium smegmatis and Mycobacterium bovis BCG. The M. smegmatis ndh mutants were highly resistant to INH and ETH, while the M. bovis BCG mutants had low-level resistance to INH and ETH. All mutants had defects in NdhII activity resulting in an increase in intracellular NADH/NAD(+) ratios. Increasing NADH levels were shown to protect InhA against inhibition by the INH-NAD adduct formed upon INH activation. We conclude that ndh mutations mediate a novel mechanism of resistance by increasing the NADH cellular concentration, which competitively inhibits the binding of INH-NAD or ETH-NAD adduct to InhA.
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PMID:Altered NADH/NAD+ ratio mediates coresistance to isoniazid and ethionamide in mycobacteria. 1567 55

The P450mor system from Mycobacterium sp. strain HE5, supposed to catalyse the hydroxylation of different N-heterocycles, is composed of three components: ferredoxin reductase (FdRmor), Fe3S4 ferredoxin (Fdmor) and cytochrome P450 (P450mor). In this study, we purified Fdmor and P450mor as recombinant proteins as well as FdRmor, which has been isolated previously. Kinetic investigations of the redox couple FdRmor/Fdmor revealed a 30-fold preference for the NADH-dependent reduction of nitroblue tetrazolium (NBT) and an absolute requirement for Fdmor in this reaction, compared with the NADH-dependent reduction of cytochrome c. The quite low Km (5.3 +/- 0.3 nm) of FdRmor for Fdmor, measured with NBT as the electron acceptor, indicated high specificity. The addition of sequences providing His-tags to the N- or C-terminus of Fdmor did not significantly alter kinetic parameters, but led to competitive background activities of these fusion proteins. Production of P450mor as an N-terminal His-tag fusion protein enabled the purification of this protein in its spectral active form, which has previously not been possible for wild-type P450mor. The proposed substrates morpholine, piperidine or pyrrolidine failed to produce substrate-binding spectra of P450mor under any conditions. Pyridine, metyrapone and different azole compounds generated type II binding spectra and the Kd values determined for these substances suggested that P450mor might have a preference for more bulky and/or hydrophobic molecules. The purified recombinant proteins FdRmor, Fdmor and P450mor were used to reconstitute the homologous P450-containing mono-oxygenase, which was shown to convert morpholine.
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PMID:Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5. 1572 Mar 89

Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa(3) system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics.
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PMID:Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs. 1576 66

We report the molecular cloning, expression and partial characterization of MT FdR, an FAD-associated flavoprotein, from Mycobacterium tuberculosis similar to the oxygenase-coupled NADH-dependent ferredoxin reductases (ONFR). We establish, through kinetic and spectral analysis, that MT FdR preferentially uses NADH as cofactor. Furthermore, MT FdR forms a complex with mycobacterial ferredoxin (MT Fdx) and MT CYP51, a cytochrome P450 (CYP) from M. tuberculosis that is similar to lanosterol 14alpha-demethylase isozymes. This reconstituted system transfers electrons from the cofactor to the heme iron of MT CYP51 and effects the demethylation of lanosterol.
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PMID:MT FdR: a ferredoxin reductase from M. tuberculosis that couples to MT CYP51. 1586 94

The peroxiredoxin AhpC from Mycobacterium tuberculosis (MtAhpC) is the foremost element of a NADH-dependent peroxidase and peroxynitrite reductase system, where it directly reduces peroxides and peroxynitrite and is in turn reduced by AhpD and other proteins. Overexpression of MtAhpC in isoniazid-resistant strains of M. tuberculosis harboring mutations in the catalase/peroxidase katG gene provides antioxidant protection and may substitute for the lost enzyme activities. We report here the crystal structure of oxidized MtAhpC trapped in an intermediate oligomeric state of its catalytic cycle. The overall structure folds into a ring-shaped hexamer of dimers instead of the usual pentamer of dimers observed in other reduced peroxiredoxins. Although the general structure of the functional dimer is similar to that of other 2-Cys peroxiredoxins, the alpha-helix containing the peroxidatic cysteine Cys61 undergoes a unique rigid-body movement to allow the formation of the disulfide bridge with the resolving cysteine Cys174. This conformational rearrangement creates a large internal cavity enclosing the active site, which might be exploited for the design of inhibitors that could block the catalytic cycle. Structural and mutagenesis evidence points to a model for the electron transfer pathway in MtAhpC that accounts for the unusual involvement of three cysteine residues in catalysis and suggests a mechanism by which MtAhpC can specifically interact with different redox partners.
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PMID:Structure and mechanism of the alkyl hydroperoxidase AhpC, a key element of the Mycobacterium tuberculosis defense system against oxidative stress. 1588 7

The increasing prevalence of tuberculosis in many areas of the world, associated with the rise in drug-resistant Mycobacterium tuberculosis (MTB) strains, presents a major threat to global health. InhA, the enoyl-ACP reductase from MTB, catalyzes the nicotinamide adenine dinucleotide (NADH)-dependent reduction of long-chain trans-2-enoyl-ACP fatty acids, an intermediate in mycolic acid biosynthesis. Mutations in the structural gene for InhA are associated with isoniazid resistance in vivo due to a reduced affinity for NADH, suggesting that the mechanism of drug resistance may be related to specific interactions between enzyme and cofactor within the NADH binding site. To compare the molecular events underlying ligand affinity in the wild-type, I21V, and I16T mutant enzymes and to identify the molecular aspects related to resistance, molecular dynamics simulations of fully solvated NADH-InhA (wild-type and mutants) were performed. Although very flexible, in the wild-type InhA-NADH complex, the NADH molecule keeps its extended conformation firmly bound to the enzyme's binding site. In the mutant complexes, the NADH pyrophosphate moiety undergoes considerable conformational changes, reducing its interactions with its binding site and probably indicating the initial phase of ligand expulsion from the cavity. This study should contribute to our understanding of specific molecular mechanisms of drug resistance, which is central to the design of more potent antimycobacterial agents for controlling tuberculosis.
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PMID:Molecular dynamics simulation studies of the wild-type, I21V, and I16T mutants of isoniazid-resistant Mycobacterium tuberculosis enoyl reductase (InhA) in complex with NADH: toward the understanding of NADH-InhA different affinities. 1590 76

Cloning and sequencing of the morABC operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by Mycobacterium sp. strain HE5. The cytochrome P450 (P450(mor)) and the Fe(3)S(4) ferredoxin (Fd(mor)), encoded by morA and morB, respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450(mor) system. Analysis of the mor operon has now revealed the gene morC, encoding the ferredoxin reductase of this morpholine monooxygenase. The genes morA, morB and morC were identical to the corresponding genes from Mycobacterium sp. strain RP1. Almost identical mor genes in Mycobacterium chlorophenolicum PCP-1, in addition to an inducible cytochrome P450, pointing to horizontal gene transfer, were now identified. No evidence for a circular or linear plasmid was found in Mycobacterium sp. strain HE5. Analysis of the downstream sequences of morC revealed differences in this gene region between Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1 on the one hand, and M. chlorophenolicum on the other hand, indicating insertions or deletions after recombination. Downstream of the mor genes, the gene orf1', encoding a putative glutamine synthetase, was identified in all studied strains. The gene morC of Mycobacterium sp. strain HE5 was heterologously expressed. The purified recombinant protein FdR(mor) was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The K(m) values of FdR(mor) for the substrate NADH (37.7 +/- 4.1 microM) and the artificial electron acceptors potassium ferricyanide (14.2 +/- 1.1 microM) and cytochrome c (28.0 +/- 3.6 microM) were measured. FdR(mor) was shown to interact functionally with its natural redox partner, the Fe(3)S(4) protein Fd(mor), and with the Fe(2)S(2) protein adrenodoxin, albeit with a much lower efficiency, but not with spinach ferredoxin. In contrast, adrenodoxin reductase, the natural redox partner of adrenodoxin, could not use Fd(mor) in activity assays. These results indicated that FdR(mor) can utilize different ferredoxins, but that Fd(mor) requires the specific NADH : ferredoxin oxidoreductase FdR(mor) from the P450(mor) system for efficient catalytic function.
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PMID:Analysis of the nearly identical morpholine monooxygenase-encoding mor genes from different Mycobacterium strains and characterization of the specific NADH : ferredoxin oxidoreductase of this cytochrome P450 system. 1607 38

Isoniazid is an antituberculosis prodrug that requires activation by the catalase-peroxidase (KatG) of Mycobacterium tuberculosis. The activated species, presumed to be an isonicotinoyl radical, couples to NADH forming an isoniazid-NADH adduct that ultimately confers antitubercular activity. We have compared the catalytic properties of three KatGs associated with isoniazid resistance (resistance mutation KatGs, (RM)KatGs: R104L, H108Q, S315T) to wild-type enzyme and two additional lab mutations (wild-type phenotype KatGs, (WTP)KatGs: WT KatG, Y229F, R418L). Neither catalase nor peroxidase activities, nor the presence/absence of the Met-Tyr-Trp cross-link (as probed by LC/MS on tryptic digests of the protein), exhibited any correlation with isoniazid resistance. The yields of isoniazid-NADH adduct formed were determined to be 1-5, 4-12, and 20-70-fold greater for the (WTP)KatGs than the (RM)KatGs for the compound I, II, and III pathways, respectively, strongly suggesting a role for oxyferrous KatG (supported by superoxide consumption measurements) that correlates with drug resistance. Stopped-flow UV-visible spectroscopic studies revealed that all KatGs were capable of forming both compound II and III intermediates. Rates of compound II decay were accelerated 4-12-fold in the presence of isoniazid (vs absence) for the (WTP)KatGs but were unaffected by the drug for the (RM)KatGs. A mechanism for isoniazid resistance which accounts for the observed reactivity for each of the compound I, II, and III intermediates is proposed and suggests that the compound III pathway may be the primary factor in determining overall isoniazid resistance by specific KatG mutants, with secondary contributions arising from the compound I and II pathways.
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PMID:Correlation between isoniazid resistance and superoxide reactivity in mycobacterium tuberculosis KatG. 1617 77

Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20 degrees C to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20 degrees C to 55 degrees C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s(-1) at 50 degrees C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.
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PMID:Gene cloning and characterization of Mycobacterium phlei flavin reductase involved in dibenzothiophene desulfurization. 1623 34

Biotransformation difference between parent strain (MF2) and mutant strain (MF96) of Mycobacterium fortuitum was observed. Biotransformation with resting cells showed that the major products of biotransformation by both parent and mutant strains are delta4-androstenedione(4AD) and testosterone(TS). Experiments with cell-free extract system showed that the proportion of 4AD/TS obtained from parent and mutant strains was almost same when enough NAD+ and NADH were supplied in this system. It was suggested that the difference of the ratio of products transformed by both strains in resting cell system may result from their different ratio of NAD+/NADH. This speculation was verified to be true by determination of the amount of NAD+ and NADH presented in both strains.
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PMID:[Mechanism study on difference of biotransformation between Mycobacterium fortuitum MF2 and MF96]. 1624 63


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