| Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase. | |
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MedLine Citation:
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PMID: 12071965 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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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Authors:
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Federico Fischer; Debora Raimondi; Alessandro Aliverti; Giuliana Zanetti |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: European journal of biochemistry / FEBS Volume: 269 ISSN: 0014-2956 ISO Abbreviation: Eur. J. Biochem. Publication Date: 2002 Jun |
Date Detail:
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Created Date: 2002-06-19 Completed Date: 2002-08-19 Revised Date: 2007-07-23 |
Medline Journal Info:
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Nlm Unique ID: 0107600 Medline TA: Eur J Biochem Country: Germany |
Other Details:
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Languages: eng Pagination: 3005-13 Citation Subset: IM |
Affiliation:
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Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Milano, Italy. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Anaerobiosis Bacterial Proteins / chemistry*, genetics, metabolism Catalysis Electron Transport / physiology Ferredoxin-NADP Reductase / chemistry*, genetics, metabolism* Ferredoxins / isolation & purification, metabolism* Flavoproteins / chemistry, metabolism* Mycobacterium tuberculosis / enzymology* Oxidation-Reduction Recombinant Proteins / chemistry, genetics, metabolism |
| Chemical | |
Reg. No./Substance:
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0/Bacterial Proteins; 0/Ferredoxins; 0/Flavoproteins; 0/Recombinant Proteins; EC 1.18.1.2/Ferredoxin-NADP Reductase |
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