cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3 beta,5 alpha-hydroxysteroids

This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues, Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified, The new cDNA hybridized with two messages of similar to 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain, The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambda gt11 cDNA library, The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of similar to 40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min(-1) for ethanol at pH 7.4 and 25 degrees C. ADH-F was stereospecific for the 3 beta,5 alpha-versus 3 beta,5 beta-hydroxysteroids. The K-m value for ethanol at pH 7.4 was 17 mM compared with 56 mu M for all-trans-retinol and 31 mu M for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.