Molecular cloning and characterization of (R)-3-hydroxybutyrate dehydrogenase from human heart.
The complete amino acid sequence of human heart (R)-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has been deduced from the nucleotide sequence of cDNA clones. This mitochondrial enzyme has an absolute and specific requirement of phosphatidylcholine for enzymic activity (allosteric activator) and is an important prototype of lipid-requiring enzymes. Despite extensive studies, the primary sequence has not been available and is now reported. The mature form of the enzyme consists of 297 amino acids (predicted M(r) of 33,117), does not appear to contain any transmembrane helices, and is homologous with the family of short-chain alcohol dehydrogenases (SC-ADH) (Persson, B., Krook, M., and Jörnvall, H. (1991) Eur. J. Biochem. 200, 537-543) (30% residue identity with human 17 beta-hydroxysteroid dehydrogenase). The first two-thirds of the enzyme includes both putative coenzyme binding and active site conserved residues and exhibits a predicted secondary structure motif (alternating alpha-helices and beta-sheet) characteristic of SC-ADH. Bovine heart peptide sequences (174 residues in nine sequences determined by microsequencing) have extensive homology (89% identical residues) with the deduced human heart sequence. The C-terminal third (Asn-194 to Arg-297) shows little sequence homology with the SC-ADH and likely contains elements that determine the substrate specificity for the enzyme including the phospholipid (phosphatidylcholine) binding site(s). Northern blot analysis identifies a 1.3-kilobase mRNA encoding the enzyme in heart tissue.
Marks
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Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029.
McIntyre
J O
JO
Duncan
T M
TM
Erdjument-Bromage
H
H
Tempst
P
P
Fleischer
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D006801:Humans; D006884:Hydroxybutyrate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006884:Hydroxybutyrate Dehydrogenase