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 method, using primers carrying the conserved nucleotide sequence among mammalian CRs, we isolated three different cDNAs (CHCR1, CHCR2 and CHCR3) which encode a unique carbonyl reductase from the Chinese hamster. The PCR products of CHCR1 and CHCR2 were clearly isolated with Bpu1102I, BspEI and XmaI restriction enzymes. The nucleotide-sequence of CHCR3 was completely different from those of CHCR1 and CHCR2. The predicted double-wound betaalphabetaalpha-structures of the CHCRs suggests the presence of a typical NADP(+)-binding motif and is similar to the corresponding region of 3alpha,20beta-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 homology to human CBR3 (>86%) and a relatively lower homology to the other CHCRs (<76%). Bacterial recombinant CHCRs showed typical carbonyl reductase activities towards 4-benzoylpyridine, 4-nitrobenzaldehyde and pyridine 4-carboxyaldehyde. These three CRs showed not only 3-keto reductase of steroids, but also 20-keto reductase. However, these CRs did not show any activity of 17-keto reductase activity. Both CHCR1 and CHCR2 have prostaglandin 9-keto reductase and 15-hydroxyprostaglandin dehydrogenase activities towards PGE(2) and PGF(2alpha) from the analyses of enzymatic reaction products. The results of Western blotting and RT-PCR suggest these CHCRs have a tissue-dependent-distribution in the Chinese hamster.
Terada
T
T
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka, Japan. terada@phs.osaka-u.ac.jp
Sugihara
Y
Y
Nakamura
K
K
Sato
R
R
Sakuma
S
S
Fujimoto
Y
Y
Fujita
T
T
Inazu
N
N
Maeda
M
M
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases