Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol

Abstract Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C28- and C29-Δ24(28)-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally the reaction proceeds in two steps via the Δ24(25)-intermediate. In the present study, we characterized the ArDWF1 gene from an EST library of Ajuga reptans var. atropurpurea hairy roots. The gene was functionally expressed in yeast T21 strain. The in vivo and in vitro study of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by gas chromatography-mass spectrometry (GC-MS) analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was unequivocally established by the incubation of the enzyme with (23,23,25-2H3,28-13C)-24-methylenecholesterol followed by 13C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ24(25)-intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes, Oryza sativa DWF1 (OsDWF1) was similarly expressed in yeast. An in vivo assay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in the Ajuga hairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.