Enzymic Synthesis of Lignin Precursors

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Abstract
A cinnamoyl-coenzyme A reductase catalyzing the NADPH-dependent reduction of substituted cinnamoyl-CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660-fold purification of the enzyme was achieved by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-100 and affinity chromatography on 5′-AMP-Sepharose. The apparent molecular weight of the reductase was found to be about 38000 on the basis of the elution volume from a Sephadex G-100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1—0.2 M citrate buffer at 30 °C. The enzyme was markedly inhibited by thiol reagents. The reductase showed a high degree of specificity for cinnamoyl-CoA esters. Feruloyl-CoA was the substrate with the lowest Km value (73 μM) and highest V (230 nkat/mg) followed by 5-hydroxyferuloyl-CoA, sinapoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA and cinnamoyl-CoA. No reaction took place with acetyl-CoA. The Km value for NADPH varied with the type of substrate. Km values of 28, 120, and 290 pM were found with feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA, respectively. The rate of reaction observed with NADH was only about 5 % of that found with NADPH. The reaction products CoASH and NADP+ inhibited the reaction. The Ki values were in the range of 0.5—1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed.

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