title: Expression and characterization of a functional canine variant of cytochrome b5 reductase.
abstract: Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.
authors.affiliation: Department of Molecular Medicine, School of Basic Biomedical Sciences, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd, MDC 007, Tampa, FL 33612, USA.
authors.forename: Glenn W
authors.initials: GW
authors.lastname: Roma
authors.forename: Louis J
authors.initials: LJ
authors.lastname: Crowley
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Barber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D042966:Cytochrome-B(5) Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D005182:Flavin-Adenine Dinucleotide / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D051381:Rats; D013057:Spectrum Analysis; D013816:Thermodynamics
chemical_list: D011725:Pyridines; D005182:Flavin-Adenine Dinucleotide; D009247:NADH, NADPH Oxidoreductases; D042966:Cytochrome-B(5) Reductase; C014227:ferricyanide reductase; D008058:Dihydrolipoamide Dehydrogenase