PLANT STEROL BIOSYNTHESIS - IDENTIFICATION AND CHARACTERIZATION OF HIGHER-PLANT DELTA(7)-STEROL C5(6)-DESATURASE

Microsomes obtained from maize seedlings catalyzed the introduction of the Delta(5)-bond into Delta(7)-sterols to yield the corresponding De lta(5,7)-sterols. Enzymatic bioassay conditions have been developed fo r the first time for Delta(7)-sterol C5(6)-desaturase in photosyntheti c organisms. The properties of the microsomal system have been studied and the kinetics of the desaturation reaction has been established. T he desaturation reaction requires molecular oxygen and NADH. Coenzyme efficiency studies indicate that NADH is more efficient that NADPH and that in the presence of NADH, NAD(+) stimulates the desaturation proc ess but cannot sustain the reaction by itself. The desaturation is str ongly inhibited by cyanide, is sensitive to 1,10-phenanthroline and to salicylhydroxamic acid, but is insensitive to carbon monoxide, sugges ting the involvement of a metal ion, presumably iron, in an enzyme-bou nd form in the desaturating system. From a series of incubations with Delta(7)-sterols and other sterol analogs, the substrate specificity f or desaturation was determined. Our data indicate the substrate select ivity of the C5(6)desaturation for 4-desmethyl-Delta(7)-sterols. Moreo ver, the results show that specificity of maize C5(6)-desaturase favor ed Delta(7)-sterols possessing a C24-methylene or ethylidene substitue nt compared to 24-ethyl-substituted Delta(7)-sterols. Finally, the res ults demonstrate directly that during plant sterol synthesis the Delta (5)-bond is introduced via the sequence Delta(7)-sterol --> Delta(5,7) -sterol --> Delta(5)-sterol. (C) 1996 Academic Press, Inc.