Role of the His57-Glu214 ionic couple located in the active site of Mycobacterium tuberculosis FprA.
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.
Pennati
Andrea
A
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.
Razeto
Adelia
A
de Rosa
Matteo
M
Pandini
Vittorio
V
Vanoni
Maria Antonietta
MA
Mattevi
Andrea
A
Coda
Alessandro
A
Aliverti
Alessandro
A
Zanetti
Giuliana
G
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D009169:Mycobacterium tuberculosis; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction
D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D018698:Glutamic Acid; D006639:Histidine; D009249:NADP; C464438:FprA protein, M tuberculosis; D009247:NADH, NADPH Oxidoreductases