Ny. Kedishvili et al., CDNA SEQUENCE AND CATALYTIC PROPERTIES OF A CHICK-EMBRYO ALCOHOL-DEHYDROGENASE THAT OXIDIZES RETINOL AND 3-BETA,5-ALPHA-HYDROXYSTEROIDS, The Journal of biological chemistry, 272(11), 1997, pp. 7494-7500
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.