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 reduction of various sterones to produce stereoselectively the corresponding 3 beta-hydroxy derivatives. Enzymatic assay conditions have been developed to characterize this reduction step and the kinetics of the microsomal system has been established. Sterone reduction shows exclusive dependence on NADPH and is inactive with NADH. It is not sensitive to the azole inhibitors pyrifenox, ketoconazole, and itraconazole nor to phenobarbital nor pyrazole. Based on these coenzyme requirements and inhibitor susceptibility, and according to the common pattern of their 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 reduction at C-3 was determined. Our data indicate particularly that 4 alpha-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 photosynthetic 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 early intermediates in the 4 alpha-monomethylsterols-4-demethylation process. (C) 1994 Academic Press, Inc.