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 dehydrogenas...

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Bibliographic Details
Published in:The Journal of biological chemistry 1997-03, Vol.272 (11), p.7494-7500
Main Authors: Kedishvili, N.Y. (Indiana University School of Medicine, Indianapolis, IN.), Gough, W.H, Chernoff, E.A.G, Hurley, T.D, Stone, C.L, Bowman, K.D, Popov, K.M, Bosron, W.F, Li, T.K
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
ADN
ALCOHOL DESHIDROGENASA
ALCOHOLES
ALCOOL
ALCOOL DESHYDROGENASE
ALDEHIDOS
ALDEHYDE
ARN MENSAJERO
ARN MESSAGER
COMPOSICION QUIMICA
COMPOSITION CHIMIQUE
Escherichia coli
ESTEROIDES
ETANOL
ETHANOL
FOIE
HIGADO
OXIDACION
OXYDATION
POLLITO
POLLO
POULET
POUSSIN
RETINOL
SECUENCIA NUCLEOTIDICA
SEQUENCE NUCLEOTIDIQUE
STEROIDE
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Summary: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 micromolar for all-trans-retinol and 31 micromolar 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
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.272.11.7494