Oleoyl-12-hydroxylase enzyme

In this study, we identified and quantified 61 molecular species of acylglycerols, 36 PC and 35 PE in castor microsomal incubations of six [ CJFA individually. The results show the following (i) 2-oleoyl-PC is actively formed as the immediate substrate of oleoyl-12-hydroxylase, a key enzyme for castor oil biosynthesis (ii) 2-ricinoleoyl-PC is formed mainly by the hydroxylation of 2-oleoyl-PC, not from the incorporation of ricinoleate into 2-ricinoleoyl-PC and (iii) 2-oleoyl-PE is not actively formed for the biosynthesis of castor oil. 

Ricinoleate has many industrial uses, but the only commercial source is castor bean, which is hazardous. It is thus desirable to produce ricinoleate in a transgenic plant. The cDNA for oleoyl-12-hydroxylase, the key enzyme in the biosynthesis of ricinoleate, has been cloned recently (1). In order to reach the 90% ricinoleate level provided by castor bean, it will require understanding of ricinoleate production, the enzymes that move it into triglyceride (TG) and the enzymes that keep oleate available as hydroxylase substrate. We have recently developed an enzyme assay method to characterize oleoyl-12-hydroxylase using the putative substrate, 1-acyl-2-oleoyl-.yn-glycero-3-phosphocholine (2). We report here the in vitro metabolism of 2-oleoyl-PC in the microsomes isolated from immature castor bean. 

It is generally accepted that oleoyl-12-hydroxylase catalyzes the reaction from 2-oleoyl-PC to 2-ricinoleoyl-PC (5). We have recently characterized oleoyl-12-hydroxylase in micro-somes from the endosperm of immature castor bean using the putative substrate, 2-[i4C]-oleoyl-PC (2). Our results support the hypothesis that the actual substrate of oleoyl-12-hydroxylase is 2-oleoyl-PC. In our previous study, the enzyme activity was measured by the radioactivity of ricinoleate after methanolysis of the total lipids from the incubation products. In this report, for the purpose of establishing the biosynthetic pathway, radio-active intact lipid metabolites of 2-[i" C]oleoyl-PC were separated by HPLC and their radioactivity quantified. 

---