EXPRESSION OF A FUNCTIONAL CANINE CYTOCHROME b5 REDUCTASE

Abstract

Genomic sequences derived from diverse eukaryotic species have greatly increased the number of putative cytochrome b5 reductase (cb5r) sequences. BLAST searches have revealed several Canis familiaris cb5r or DIA1 gene products. However, alignments with known cb5r sequences indicated structural anomalies, including N-terminal and internal sequence variations. To confirm the canine sequence, we utilized PCR primers derived from a mammalian consensus sequence to clone and express the functional protein from beagle lung tissue. The canine cb5r cDNA sequence comprised 906 bp coding for 300 amino acid residues with 93% and 87% sequence identity to both human and rat cb5r’s, respectively, and retained all the characteristic amino acid sequence motifs associated with members of the flavoprotein transhydrogenase superfamily. Expression of the soluble canine cb5r diaphorase domain (I33-F300) resulted in a FAD-containing protein of mass (m/z) 31,505 (31,522 calc.) with spectroscopic properties identical to other mammalian cb5r’s, NADH:ferricyanide reductase activity (kcat=767 s−1, Km=7 μM NADH), a marked specificity for NADH vs. NADPH, a redox potential (n=2) for the FAD prosthetic group of −274 mV and binding constants for H4NAD (67 μM) and NAD+ (787 μM). These results identify the first correct sequence for C. familiaris cb5r and indicate a protein structurally and functionally comparable to other mammalian variants (supported by NIH).