UMLS:C0026918 - FACTA Search

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Query: UMLS:C0026918 (Mycobacterium)
52,428 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD-NADPH and FADH2-NADP+) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabilization of the one-electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe-2S] iron-sulfur proteins, either adrenodoxin or plant ferredoxin. More interestingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferredoxin purified from M. smegmatis were determined. A Km value of 30 nm and a specificity constant of 110 microM(-1) x s(-1) (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH-ferredoxin oxidoreductase.
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PMID:Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase. 1207 65

The genome sequence of the pathogenic bacterium Mycobacterium tuberculosis revealed numerous cytochrome P450 enzymes, which require accessory redox enzymes for catalytic function (ferredoxin reductase and ferredoxin). The most likely ferredoxin reductase is encoded by fprA, and its structure resembles eukaryotic adrenodoxin reductases. We have cloned, expressed and purified the flavoenzyme product of the fprA gene in Escherichia coli. FprA reduces various electron acceptors using either NADPH or NADH as the electron donor, but discriminates in favour of NADPH (apparent K (m) for NADH=50.6+/-3.1 microM; NADPH=4.1+/-0.3 microM from ferricyanide reduction experiments). Stopped-flow studies of reduction of the FprA FAD by NADPH demonstrate increased flavin reduction rate at low NADPH concentration (<200 microM), consistent with the presence of a second, kinetically distinct and inhibitory, pyridine nucleotide-binding site, similar to that identified in human cytochrome P450 reductase [Gutierrez, Lian, Wolf, Scrutton and Roberts (2001) Biochemistry 40, 1964-1975]. Flavin reduction by NADH is slower than with NADPH and displays hyperbolic dependence on NADH concentration [maximal reduction rate ( k (red))=25.4+/-0.7 s(-1), apparent K (d)=42.9+/-4.6 microM]. Flavin reoxidation by molecular oxygen is more rapid for NADH-reduced enzyme. Reductive titrations show that the enzyme forms a species with spectral characteristics typical of a neutral (blue) FAD semiquinone only on reduction with NADPH, consistent with EPR studies. The second order dependence of semiquinone formation on the concentration of FprA indicates a disproportionation reaction involving oxidized and two-electron-reduced FprA. Titration of FprA with dithionite converts oxidized FAD into the hydroquinone form; the flavin semiquinone is not populated under these conditions. The midpoint reduction potential for the two electron couple is -235+/-5 mV (versus the normal hydrogen electrode), similar to that for adrenodoxin reductase (-274 mV). Our data provide a thermodynamic and transient kinetic framework for catalysis by FprA, and complement recent spectrophotometric and steady-state studies of the enzyme [Fischer, Raimondi, Aliverti and Zanetti (2002) Eur. J. Biochem. 269, 3005-3013].
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PMID:Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA. 1261 97

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

Mycobacterium tuberculosis FprA is a NADPH-ferredoxin reductase, functionally and structurally similar to the mammalian adrenodoxin reductase. It is presumably involved in supplying electrons to one or more of the pathogen's cytochrome P450s through reduced ferredoxins. It has been proposed on the basis of crystallographic data (Bossi, R. T., et al. (2002) Biochemistry 41, 8807-8818) that the highly conserved His57 and Glu214 whose side chains are H-bonded are involved in catalysis. Both residues were individually changed to nonionizable amino acyl residues through site-directed mutagenesis. Steady-state kinetics showed that the role of Glu214 in catalysis is negligible. On the contrary, the substitutions of His57 markedly impaired the catalytic efficiency of FprA for ferredoxin in the physiological reaction. Furthemore, they decreased the k(cat)/K(m) value for NADPH in the ferricyanide reduction. Rapid-reaction (stopped-flow) kinetic analysis of the isolated reductive half-reaction of wild-type and His57Gln forms of FprA with NADPH and NADH allowed a detailed description of the mechanism of enzyme-bound FAD reduction, with the identification of the intermediates involved. The His57Gln mutation caused a 6-fold decrease in the rate of hydride transfer from either NADPH or NADH to the enzyme-bound FAD cofactor. The 3D structure of FprA-H57Q, obtained at 1.8 A resolution, explains the inefficient hydride transfer of the mutant in terms of a suboptimal geometry of the nicotinamide-isoalloxazine interaction in the active site. These data demonstrate the role of His57 in the correct binding of NADPH to FprA for the subsequent steps of the catalytic cycle to proceed at a high rate.
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PMID:Role of the His57-Glu214 ionic couple located in the active site of Mycobacterium tuberculosis FprA. 1684 14

Mycobacterium smegmatis ferredoxin FdxA, which has an orthologue ferredoxin in Mycobacterium tuberculosis, FdxC, contains both one [3Fe-4S] and one [4Fe-4S] cluster. M. smegmatis FdxA has been shown to be a preferred ferredoxin substrate of FprA [F. Fischer, D. Raimondi, A. Aliverti, G. Zanetti, Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase, Eur. J. Biochem. 269 (2002) 3005-3013], an adrenodoxin reductase-like flavoprotein of M. tuberculosis, suggesting that M. tuberculosis FdxC could be the physiological partner of the enzyme in providing reducing power to the cytochromes P450. We report here the crystal structure of FdxA at 1.6A resolution (R(factor) 16.5%, R(free) 20.2%). Besides providing an insight on protein architecture for this 106-residue ferredoxin, our crystallographic investigation highlights lability of the [4Fe-4S] center, which is shown to loose a Fe atom during crystal growth. Due to their high similarity (87% sequence identity), the structure here reported can be considered a valuable model for M. tuberculosis FdxC, thus representing a step forward in the study of the complex mycobacterial redox pathways.
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PMID:The crystal structure of FdxA, a 7Fe ferredoxin from Mycobacterium smegmatis. 1757 75

We have previously shown that Mycobacterium tuberculosis FprA, an NADPH-ferredoxin reductase homologous to mammalian adrenodoxin reductase, promotes the oxidation of NADP(+) to its 4-oxo derivative 3-carboxamide-4-pyridone adenine dinucleotide phosphate [Bossi RT, Aliverti A, Raimondi D, Fischer F, Zanetti G, Ferrari D, Tahallah N, Maier CS, Heck AJ, Rizzi M et al. (2002) Biochemistry41, 8807-8818]. Here, we provide a detailed study of this unusual enzyme reaction, showing that it occurs at a very slow rate (0.14 h(-1)), requires the participation of the enzyme-bound FAD, and is regiospecific in affecting only the C4 of the NADP nicotinamide ring. By protein engineering, we excluded the involvement in catalysis of residues Glu214 and His57, previously suggested to be implicated on the basis of their localization in the three-dimensional structure of the enzyme. Our results substantiate a catalytic mechanism for 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation in which the initial and rate-determining step is the nucleophilic attack of the nicotinamide moiety by an active site water molecule. Whereas plant-type ferredoxin reductases were unable to oxidize NADP(+), the mammalian adrenodoxin reductase also catalyzed this unusual reaction. Thus, the 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation reaction seems to be a peculiar feature of the mitochondrial type of ferredoxin reductases, possibly reflecting conserved properties of their active sites. Furthermore, we showed that 3-carboxamide-4-pyridone adenine dinucleotide phosphate is good ligand and a competitive inhibitor of various dehydrogenases, making this nucleotide analog a useful tool for the characterization of the cosubstrate-binding site of NADPH-dependent enzymes.
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PMID:Enzymatic oxidation of NADP+ to its 4-oxo derivative is a side-reaction displayed only by the adrenodoxin reductase type of ferredoxin-NADP+ reductases. 1763 83

Mtb (Mycobacterium tuberculosis) FprA (flavoprotein reductase A) is an NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports the catalytic function of Mtb cytochrome P450s. Trp(359), proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp(359) is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define the role of Trp(359). W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone formed in wild-type NADPH-reduced FprA was destabilized in the W359A/H mutants, which also had more positive FAD midpoint reduction potentials (-168/-181 mV respectively, versus the standard hydrogen electrode, compared with -230 mV for wild-type FprA). The W359A/H mutants had lower ferricyanide reductase k(cat) and NAD(P)H K(m) values, but this led to improvements in catalytic efficiency (k(cat)/K(m)) with NADH as reducing coenzyme (9.6/18.8 muM(-1).min(-1) respectively, compared with 5.7 muM(-1).min(-1) for wild-type FprA). Stopped-flow spectroscopy revealed NAD(P)H-dependent FAD reduction as rate-limiting in steady-state catalysis, and to be retarded in mutants (e.g. limiting rate constants for NADH-dependent FAD reduction were 25.4 s(-1) for wild-type FprA and 4.8 s(-1)/13.4 s(-1) for W359A/H mutants). Diminished mutant FAD content (particularly in W359H FprA) highlighted the importance of Trp(359) for flavin stability. The results demonstrate that the conserved Trp(359) is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.
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PMID:Trp(359) regulates flavin thermodynamics and coenzyme selectivity in Mycobacterium tuberculosis FprA. 1823 73

Despite a number of studies, the formation of the Michaelis complexes between ferredoxin-NADP (+) reductases and NADP(H) eluded detailed investigations by rapid kinetic techniques because of their high formation rates. Moreover, the reversible nature of the reaction of hydride ion transfer between these enzymes and NADPH prevented the obtainment of reliable estimates of the rate constant of the hydride transfer step. Here we show that by working at a high salt concentration, the mechanism of the reaction with NADPH of FprA, a Mycobacterium tuberculosis homologue of adrenodoxin reductase, is greatly simplified, making it amenable to investigation by rapid reaction techniques. The approach presented herein allowed for the first time the observation of the formation of the Michaelis complex between an adrenodoxin reductase-like enzyme and NADPH, and the determination of the related rate constants for association and dissociation. Furthermore, the rate constant for the reaction of hydride ion transfer between NADPH and FAD could be unambiguously assessed. It is proposed that the approach described should be applicable to other ferredoxin reductase enzymes, providing a valuable experimental tool for the study of their kinetic properties.
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PMID:Effect of salt and pH on the reductive half-reaction of Mycobacterium tuberculosis FprA with NADPH. 1829 30

Mycobacterium tuberculosis FprA (flavoprotein reductase A) is an NAD(P)H- and FAD-binding reductase that is structurally/evolutionarily related to adrenodoxin reductase. Structural analysis implicates Arg(199) and Arg(200) in interactions with the NADP(H) 2'-phosphate group. R199A, R200A and R199A/R200A mutants were characterized to explore the roles of these basic residues. All mutations abolished neutral FAD semiquinone stabilization in the NADPH-reduced enzyme, owing to weakened NADPH affinity. Instead, FAD hydroquinone was formed in all mutants, and each displayed substantially enhanced autooxidation rates (20-40-fold) compared with NADPH-reduced WT (wild-type) FprA. Steady-state ferricyanide reduction studies revealed diminished NADPH affinity (higher K(m) values), but lower NADH K(m) values. Despite a lowered k(cat), the R199A/R200A mutant exhibited a 200-fold coenzyme specificity switch towards NADH, although substrate inhibition was observed at high NADH concentrations (K(i)=250 microM). Stopped-flow FAD reduction studies confirmed substantially increased NADPH K(d) values, although the limiting flavin reduction rate constant was similar in all mutants. The R199A mutation abolished electron transfer between hydroquinone FprA and NADP+, while this reaction progressed (via an FADH(2)-NADP+ charge-transfer intermediate) for R200A FprA, albeit more slowly (k(lim)=58.1 s(-1) compared with >300 s(-1)) than in WT. All mutations caused positive shifts in FAD potential (approximately 40-65 mV). Binding of an NADPH analogue (tetrahydro-NADP) induced negative shifts in potential ( approximately 30-40 mV) only for variants with the R200A mutation, indicating distinctive effects of Arg(199)/Arg(200) on coenzyme binding mode and FAD potential. Collectively, these data reveal important roles for the phylogenetically conserved arginines in controlling FprA FAD environment, thermodynamics, coenzyme selectivity and reactivity.
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PMID:Characterization of coenzyme binding and selectivity determinants in Mycobacterium tuberculosis flavoprotein reductase A: analysis of Arg(199) and Arg(200) mutants at the NADP(H) 2'-phosphate binding site. 1876 89