cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3beta,5alpha-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 approximately 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 lambdagt11 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 approximately 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 3beta,5alpha- versus 3beta,5beta-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM 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.
Kedishvili
N Y
NY
Department of Biochemistry and Molecular Biology, Purdue University, Indianapolis, Indiana 46202-5122, USA.
Gough
W H
WH
Chernoff
E A
EA
Hurley
T D
TD
Stone
C L
CL
Bowman
K D
KD
Popov
K M
KM
Bosron
W F
WF
Li
T K
TK
D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002642:Chick Embryo; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics*; D006914:Hydroxysteroids / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D014801:Vitamin A / Q000378:metabolism*
D018076:DNA, Complementary; D006914:Hydroxysteroids; D014801:Vitamin A; D000426:Alcohol Dehydrogenase