Clarification of cinnamoyl co-enzyme a reductase catalysis in monolignol biosynthesis of aspen
Clarification of cinnamoyl co-enzyme a reductase catalysis in monolignol biosynthesis of aspen
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DOI：
10.1093/pcp/pci120
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发表时间：
2005-07-01
影响因子：
4.9
通讯作者：
Chiang, VL
中科院分区：
文献类型：
--
作者：
Li, LG;Cheng, XF;Chiang, VL
肉桂酰基辅酶A还原酶（CCR）是参与单脂醇生物合成的关键酶之一，被认为可以催化几种肉桂酰基辅酶A酯转化为相应的肉桂醛。然而，尚不清楚哪种肉桂酰辅酶a酯在单脂醇生物合成中被代谢。从发育中的白杨木质部组织中克隆了一个木质部特异性的CCR cDNA。通过大肠杆菌表达系统制备了重组CCR蛋白，并进行了电泳纯化。通过直接结构确证和液相色谱-质谱联用系统对反应产物进行定量分析，表征了CCR的生化特性。酶动力学表明，CCR选择性催化从5个肉桂酰辅酶a酯混合物中还原阿魏酰辅酶a。此外，阿魏酰辅酶a对其他肉桂酰辅酶a酯的CCR催化表现出很强的竞争性抑制作用。重要的是，当CCR与咖啡酰辅酶a o -甲基转移酶（CCoAOMT）偶联催化底物咖啡酰辅酶a酯时，形成了针叶醛，这表明CCoAOMT和CCR是相邻的酶。然而，体外实验结果也表明，这两种相邻酶介导的反应需要不同的pH环境，这表明CCR和CCoAOMT可能需要区隔化才能在体内正常发挥作用。在毛藻基因组中鉴定出8个CCR同源基因，它们的表达谱表明它们的功能可能存在差异。
Cinnamoyl co-enzyme A reductase (CCR), one of the key enzymes involved in the biosynthesis of monolignols, has been thought to catalyze the conversion of several cinnamoyl-CoA esters to their respective cinnamaldehydes. However, it is unclear which cinnamoyl-CoA ester is metabolized for monolignol biosynthesis. A xylem-specific CCR cDNA was cloned from aspen (Populus trentuloides) developing xylem tissue. The recombinant CCR protein was produced through an Escherichia coli expression system and purified to electrophoretic homogeneity. The biochemical properties of CCR were characterized through direct structural corroboration and quantitative analysis of the reaction products using a liquid chromatography-mass spectrometry system. The enzyme kinetics demonstrated that CCR selectively catalyzed the reduction of feruloyl-CoA from a mixture of five cinnamoyl CoA esters. Furthermore, feruloyl-CoA showed a strong competitive inhibition of the CCR catalysis of other cinnamoyl CoA esters. Importantly, when CCR was coupled with caffeoyl-CoA O-methyltransferase (CCoAOMT) to catalyze the substrate caffeoyl-CoA ester, coniferaldehyde was formed, suggesting that CCoAOMT and CCR are neighboring enzymes. However, the in vitro results also revealed that the reactions mediated by these two neighboring enzymes require different pH environments, indicating that compartmentalization is probably needed for CCR and CCoAOMT to function properly in vivo. Eight CCR homologous genes were identified in the P trichocarpa genome and their expression profiling suggests that they may function differentially.