Microsomal Δ8,14‐sterol Δ14‐reductase in higher plants

Abstract

An enzymatic assay for .DELTA.8,14-sterol .DELTA.14-reductase, an enzyme involved in sterol biosynthesis, has been developed for the first time in higher plants. The properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics and substrate specificity. This enzymatic double-bound reduction is thought to proceed through an electronophilic addition mechanism, involving a C14 putative carbonium ion high-energy intermediate. Using this in vitro assay, ammonium and iminium analogoues of this cationic intermediate were shown to be potent inhibitors of the reduction reaction. Thus, compounds of the N-alkyl-8-aza-4.alpha.,10-dimethyl-trans-decal-3.beta.-ol series strongly inhibited sterol reductase (I50 = 0.07-4 .mu.M) (I50/Km = 10-4-10-3), as did the antimycotic agent 15-azasterol (I50 = 0.03 .mu.M); all of these compounds act as reaction-intermediate analogues of the proposed C14 carbonium ion intermediate. Moreover, the in vitro inhibiton of the plant sterol reductase by a series of ammonium-ion-containing fungicides was demonstrated. The reative specificity of these different series of inhibitors toward cycloeucalenol-obtusifoliol isomerase, .DELTA.8.fwdarw..DELTA.7-sterol isomerase and .DELTA.8,14-sterol .DELTA.14-reductase, was directly studied.