Plant Sterol Biosynthesis: Identification and Characterization of Higher Plant Δ7-Sterol C5(6)-Desaturase

Microsomes obtained from maize seedlings catalyzed the introduction of the Δ5-bond into Δ7-sterols to yield the corresponding Δ5,7-sterols. Enzymatic bioassay conditions have been developed for the first time for Δ7-sterol C5(6)-desaturase in photosynthetic organisms. The properties of the microsomal system have been studied and the kinetics of the desaturation reaction has been established. The 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 process but cannot sustain the reaction by itself. The desaturation is strongly inhibited by cyanide, is sensitive to 1,10-phenanthroline and to salicylhydroxamic acid, but is insensitive to carbon monoxide, suggesting the involvement of a metal ion, presumably iron, in an enzyme-bound form in the desaturating system. From a series of incubations with Δ7-sterols and other sterol analogs, the substrate specificity for desaturation was determined. Our data indicate the substrate selectivity of the C5(6)-desaturation for 4-desmethyl-Δ7-sterols. Moreover, the results show that specificity of maize C5(6)-desaturase favored Δ7-sterols possessing a C24-methylene or ethylidene substituent compared to 24-ethyl-substituted Δ7-sterols. Finally, the results demonstrate directly that during plant sterol synthesis the Δ5-bond is introduced via the sequence Δ7-sterol → Δ5,7-sterol → Δ5-sterol.