PLANT STEROL BIOSYNTHESIS - IDENTIFICATION OF A NADPH DEPENDENT STERONE REDUCTASE INVOLVED IN STEROL-4 DEMETHYLATION

Microsomes obtained from maize embryos were shown to catalyze the redu ction of various sterones to produce stereoselectively the correspondi ng 3 beta-hydroxy derivatives. Enzymatic assay conditions have been de veloped to characterize this reduction step and the kinetics of the mi crosomal system has been established. Sterone reduction shows exclusiv e dependence on NADPH and is inactive with NADH. It is not sensitive t o the azole inhibitors pyrifenox, ketoconazole, and itraconazole nor t o phenobarbital nor pyrazole. Based on these coenzyme requirements and inhibitor susceptibility, and according to the common pattern of thei r classification, the maize microsomal sterone-reducing enzyme belongs to the family of ketone reductases. From a series of incubations with natural or synthetic sterones, the substrate specificity of the reduc tion at C-3 was determined. Our data indicate particularly that 4 alph a-methyl-9 beta,19-cyclo-C-30-sterones and 4-desmethyl-Delta(7)-C-27- or C-30-sterones are preferentially reduced, while 4,4-dimethyl-C-30- or C-31-sterones react poorly. The results support the conclusion that the reductase activity identified is a constitutive component of the microsomal sterol 4-demethylation complex recently identified in photo synthetic organisms (S. Pascal ct al., 1993, J. Biol. Chem. 268, 11639 ). They are consistent with the conclusion that 4 alpha-methylsterones are demethylation products of 4,4-gem-dimethylsterols rather than ear ly intermediates in the 4 alpha-monomethylsterols-4-demethylation proc ess. (C) 1994 Academic Press, Inc.