cDNA cloning, expression, and mutagenesis study of leukotriene B4 12-hydroxydehydrogenase.
Leukotriene B4 12-hydroxydehydrogenase catalyzes the conversion of leukotriene B4 into its biologically less active metabolite, 12-oxo-leukotriene B4. This is an initial and key step of metabolic inactivation of leukotriene B4 in various tissues other than leukocytes. Here we report the cDNA cloning for porcine and human enzymes from kidney cDNA libraries. A full-length cDNA of the porcine enzyme contains an open reading frame consisting of 987 base pairs, corresponding to 329 amino acids. The human enzyme showed a 97.1% homology with the porcine enzyme. Northern blotting of human tissues revealed its high expression in the kidney, liver, and intestine but not in leukocytes. The porcine enzyme was expressed as a glutathione S-transferase fusion protein in Escherichia coli, which exhibited similar characteristics with the native enzyme. Because the enzymes have a homology, in part, with NAD(P)(+)-dependent alcohol dehydrogenases, a site-directed mutagenesis study was carried out. We found that three glycines at 152, 155, and 166 have crucial roles in the enzyme activity, possibly by producing an NADP+ binding pocket.
Yokomizo
T
T
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Ogawa
Y
Y
Uozumi
N
N
Kume
K
K
Izumi
T
T
Shimizu
T
T
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine
D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D009249:NADP; D000429:Alcohol Oxidoreductases; C082646:leukotriene B4 12-hydroxydehydrogenase; D005982:Glutathione Transferase