Resolution and purification of an aldehyde-generating and an alcohol-generating fatty acyl-CoA reductase from pea leaves (Pisum sativum L.).

Abstract Higher plant tissues produce both wax esters generated from fatty alcohols and hydrocarbons generated from fatty aldehydes. If two different reductases are responsible for the synthesis of aldehydes and alcohols, both types of reductases may be present in such tissues. To test for this possibility, pea leaves, known to produce both types of wax components, were examined. Subcellular fractionation showed that acyl-CoA reductase activities were localized mainly in the microsomal fraction. Fatty aldehyde formation was rectilinear for 30 min and subsequently decreased, whereas fatty alcohol formation remained linear for 2 h. The two activities in the microsomes were differently affected by pH; alcohol formation was optimal between pH 5 and pH 6, whereas aldehyde formation was optimal at around pH 7.5. With solubilized microsomes, protein concentration dependence of alcohol formation showed a sigmoidal pattern, possibly suggesting inhibition by hexadecanoyl-CoA at low protein concentrations. Bovine serum albumin (BSA) enhanced alcohol formation. In contrast, the aldehyde generation showed a typical protein concentration dependence, and BSA severely inhibited aldehyde generation. Phosphatidylcholine showed over twofold stimulation for alcohol formation, whereas aldehyde formation was only slightly stimulated. All of this biochemical evidence suggested the presence of two different reductases. Confirming this hypothesis, an aldehyde-generating and an alcohol-generating reductase were resolved from the solubilized microsomal proteins using Blue A agarose, gel filtration, and hexadecanoyl-CoA affinity chromatography. SDS–PAGE of the purified proteins showed that the alcohol-generating enzyme was a 58-kDa protein and the aldehyde-forming one was a 28-kDa protein. It is proposed that two different elongating systems are functionally coupled to the alcohol-generating and aldehyde-generating reductases, which in turn are coupled to the transacylase to produce wax esters and to the decarbonylase to produce hydrocarbons, respectively.