CDNA CLONING, EXPRESSION, AND MUTAGENESIS STUDY OF LEUKOTRIENE B-4 12-HYDROXYDEHYDROGENASE

Leukotriene B-4 12-hydroxydehydrogenase catalyzes the conversion of le ukotriene B-4 into its biologically less active metabolite, 12-oxo-leu kotriene B-4, This is an initial and key step of metabolic inactivatio n of leukotriene B-4 in various tissues other than leukocytes, Here we report the cDNA cloning for porcine and human enzymes from kidney cDN A libraries. A full-length cDNA of the porcine enzyme contains an open reading frame consisting of 987 base pairs, corresponding to 329 amin o acids. The human enzyme showed a 97.1% homology with the porcine enz yme. Northern blotting of human tissues revealed its high expression i n 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 nat ive 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 ha ve crucial roles in the enzyme activity, possibly by producing an NADP (+) binding pocket.