Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA

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 resembl...

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Bibliographic Details
Published in:Biochemical journal 2003-06, Vol.372 (Pt 2), p.317-327
Main Authors: McLean, Kirsty J, Scrutton, Nigel S, Munro, Andrew W
Format: Article
Language:English
Subjects:
Binding Sites
Catalytic Domain
Circular Dichroism
Cloning, Molecular
Electron Spin Resonance Spectroscopy
Electron Transport - physiology
Escherichia coli - enzymology
Ferredoxin-NADP Reductase - chemistry
Ferredoxin-NADP Reductase - genetics
Ferredoxins - metabolism
Ferricyanides - metabolism
Flavin-Adenine Dinucleotide - metabolism
Kinetics
Mycobacterium tuberculosis
Mycobacterium tuberculosis - enzymology
NAD - metabolism
NADH, NADPH Oxidoreductases - chemistry
NADH, NADPH Oxidoreductases - genetics
NADP - metabolism
Oxidation-Reduction
Oxidoreductases - chemistry
Oxidoreductases - genetics
Protein Binding
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Spectrophotometry, Ultraviolet
Thermodynamics
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Summary: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 (
ISSN:0264-6021
1470-8728
DOI:10.1042/bj20021692