Characterization of multiple Chinese hamster carbonyl reductases

Carbonyl reductase (CR) is an enzyme which can catalyze the oxidoreduction of Various carbonyl compounds in the presence of NAD(P)H. With the PCR meth od, using primers carrying the conserved nucleotide sequence among mammalia n CRs, we isolated three different cDNAs (CHCR1, CHCR2 and CHCR3) which enc ode a unique carbonyl reductase from the Chinese hamster. The PCR products of CHCR1 and CHCR2 were clearly isolated with Bpu 11021, BspEI and XmaI res triction enzymes. The nucleotide-sequence of CHCR3 was completely different from those of CHCR1 and CHCR2. The predicted double-wound beta alpha beta alpha -structures of the CHCRs suggests the presence of a typical NADP(+)-b inding motif and is similar to the corresponding region of 3 alpha ,20 beta -hydroxysteroid dehydrogenase and mouse lung tetrameric carbonyl reductase . The deduced amino acid sequence of CHCR1 showed a high homology to CHCR2 (> 96%) and the other mammalian CRs (> 81%). However, CHCR3 showed a high h omology to human CBR3 (> 86%) and a relatively lower homology to the other CHCRs (< 76%). Bacterial recombinant CHCRs showed typical carbonyl reductas e activities towards 4-benzoylpyridine, 4-nitrobenzaldehyde and pyridine 4- carboxyaldehyde. These three CRs showed not only 3-keto reductase of steroi ds, but also 20-keto reductase. However, these CRs did not show any activit y of 17-keto reductase activity. Both CHCR1 and CHCR2 have prostaglandin 9- keto reductase and 15-hydroxyprostaglandin dehydrogenase activities towards PGE(2) and PGF(2<alpha>) from the analyses of enzymatic reaction products. The results of Western blotting and RT-PCR suggest these CHCRs have a tiss ue-dependent-distribution in the Chinese hamster. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.