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-contai ning alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues, Degenerate oligonucleotide primers were us ed to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDN A fragments that encode an unknown putative alcohol dehydrogenase as w ell as the class III alcohol dehydrogenase were identified, The new cD NA hybridized with two messages of similar to 2 and 3 kilobase pairs i n the adult chicken liver but not in the adult heart, muscle, testis, or brain, The corresponding complete cDNA clones with a total length o f 1390 base pairs were isolated from a chicken liver lambda gt11 cDNA library, The open reading frame encoded a 375-amino acid polypeptide t hat exhibited 67 and 68% sequence identity with chicken class I and II I alcohol dehydrogenases, respectively, and had lower identity with ma mmalian 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 act ivity 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 be ta-hydroxysteroids. The K-m value for ethanol at pH 7.4 was 17 mM comp ared with 56 mu M for all-trans-retinol and 31 mu M for epiandrosteron e. Antiserum against ADH-F recognized corresponding protein in the chi cken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.