Li L, Popko J L, Umezawa T, Chiang V L
Plant Biotechnology Research Center, School of Forestry and Wood Products, Michigan Technological University, Houghton, Michigan 49931, USA.
J Biol Chem. 2000 Mar 3;275(9):6537-45. doi: 10.1074/jbc.275.9.6537.
S-Adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT, EC 2.1.1.6) has traditionally been thought to catalyze the methylation of caffeate and 5- hydroxyferulate for the biosynthesis of syringyl monolignol, a lignin constituent of angiosperm wood that enables efficient lignin degradation for cellulose production. However, recent recognition that coniferyl aldehyde prevents 5-hydroxyferulate biosynthesis in lignifying tissue, and that the hydroxylated form of coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, is an alternative COMT substrate, demands a re-evaluation of the role of COMT during monolignol biosynthesis. Based on recombinant aspen (Populus tremuloides) COMT enzyme kinetics coupled with mass spectrometry analysis, this study establishes for the first time that COMT is in fact a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT), and that 5-hydroxyconiferyl aldehyde is both the preferred AldOMT substrate and an inhibitor of caffeate and 5-hydroxyferulate methylation, as measured by K(m) and K(i) values. 5-Hydroxyconiferyl aldehyde also inhibited the caffeate and 5-hydroxyferulate methylation activities of xylem proteins from various angiosperm tree species. The evidence that syringyl monolignol biosynthesis is independent of caffeate and 5-hydroxyferulate methylation supports our previous discovery that coniferyl aldehyde prevents ferulate 5-hydroxylation and at the same time ensures a coniferyl aldehyde 5-hydroxylase (CAld5H)-mediated biosynthesis of 5-hydroxyconiferyl aldehyde. Together, our results provide conclusive evidence for the presence of a CAld5H/AldOMT-catalyzed coniferyl aldehyde 5-hydroxylation/methylation pathway that directs syringyl monolignol biosynthesis in angiosperms.
S-腺苷-L-甲硫氨酸依赖性咖啡酸O-甲基转移酶（COMT，EC 2.1.1.6）传统上被认为催化咖啡酸和5-羟基阿魏酸的甲基化反应，以生物合成紫丁香基单木酚，这是被子植物木材木质素的一种成分，有助于高效降解木质素以生产纤维素。然而，最近人们认识到松柏醛会阻止木质化组织中5-羟基阿魏酸的生物合成，并且松柏醛的羟基化形式5-羟基松柏醛是COMT的另一种底物，这就需要重新评估COMT在单木酚生物合成过程中的作用。基于重组白杨（颤杨）COMT酶动力学结合质谱分析，本研究首次确定COMT实际上是一种5-羟基松柏醛O-甲基转移酶（AldOMT），并且5-羟基松柏醛既是AldOMT的首选底物，也是咖啡酸和5-羟基阿魏酸甲基化反应的抑制剂，这是通过米氏常数（K(m)）和抑制常数（K(i)）值测定得出的。5-羟基松柏醛还抑制了来自各种被子植物树种木质部蛋白质的咖啡酸和5-羟基阿魏酸甲基化活性。紫丁香基单木酚生物合成独立于咖啡酸和5-羟基阿魏酸甲基化的证据支持了我们之前的发现，即松柏醛会阻止阿魏酸的5-羟基化反应，同时确保由松柏醛5-羟化酶（CAld5H）介导的5-羟基松柏醛的生物合成。总之，我们的结果为存在一条由CAld5H/AldOMT催化的松柏醛5-羟基化/甲基化途径提供了确凿证据，该途径指导被子植物中紫丁香基单木酚的生物合成。
