Enzymic synthesis of lignin precursors. Purification and properties of a cinnamoyl-CoA: NADPH reductase from cell suspension cultures of soybean (Glycinemax).
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 38 000 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 degrees 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 muM) and highest V (230 nkat/mg) followed by 5-hydroxy-feruloyl-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 muM 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.
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D002934:Cinnamates / Q000378:metabolism; D003065:Coenzyme A; D066298:In Vitro Techniques; D008031:Lignin / Q000096:biosynthesis*; D008970:Molecular Weight; D009249:NADP; D013025:Glycine max
D002934:Cinnamates; D009249:NADP; D008031:Lignin; D000445:Aldehyde Oxidoreductases; D003065:Coenzyme A