Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR
Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR
复制标题
毛白杨肉桂酰辅酶A还原酶（CCR）基因家族的表征揭示了CCR的酶活性位点和进化
DOI：
10.1007/s00425-016-2591-6
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发表时间：
2017-01-01
期刊：
影响因子：
4.3
通讯作者：
Gai, Ying
中科院分区：
文献类型：
--
作者：
Chao, Nan;Li, Ning;Gai, Ying
从毛白杨（Populus tomentosa）中克隆了2个肉桂酰辅酶A还原酶（Cinnamoyl-coenzyme A reductases，CCR）基因，并通过序列差异分析、分子模拟和定点突变等方法对CCR的活性位点进行了鉴定。本研究克隆了11个毛白杨CCR或CCR样蛋白基因。纯化的重组毛白杨（Pto）CCR和PtoCCR样蛋白的酶促测定表明，只有PtoCCR 1和PtoCCR 7对羟基肉桂酰辅酶A酯具有可检测的活性。PtoCCR 1对阿魏酰辅酶A具有特异性，对任何其他羟基肉桂酰辅酶A酯均未检测到活性。然而，PtoCCR 7催化对香豆酰-CoA、咖啡酰-CoA、阿魏酰-CoA和芥子酰-CoA，偏好阿魏酰-CoA。定点突变的选定的氨基酸之间的分歧PtoCCR 1和7，结合建模和对接，表明A132 CCR 7结合催化三联体可能包括催化中心。在CCR 7中，L192、F155和H208被鉴定为底物结合位点，并且这些氨基酸的定点突变显示出相对于阿魏酰-CoA和芥子酰-CoA的催化效率的明显变化。突变体F155 Y对芥子酰辅酶A的催化效率高于野生型PtoCCR 7。最后，最近的基因组重复事件提供了CCR分歧的基础。本研究进一步明确了CCRs的活性位点和CCRs在陆生植物中的进化过程。
Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Populus tomentosa were cloned and studied and active sites in CCRs were further identified based on sequence divergence, molecular simulation, and site-directed mutants.Cinnamoyl-coenzyme A (CoA) reductase (CCR) is the first committed gene in the lignin-specific pathway and plays a role in the lignin biosynthesis pathway. In this study, we cloned 11 genes encoding CCR or CCR-like proteins in Populus tomentosa. An enzymatic assay of the purified recombinant P. tomentosa (Pto) CCR and PtoCCR-like proteins indicated that only PtoCCR1 and PtoCCR7 had detectable activities toward hydroxycinnamoyl-CoA esters. PtoCCR1 exhibited specificity for feruloyl-CoA, with no detectable activity for any other hydroxycinnamoyl-CoA esters. However, PtoCCR7 catalyzed p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA with a preference for feruloyl-CoA. Site-directed mutations of selected amino acids divergent between PtoCCR1 and 7, combined with modeling and docking, showed that A132 in CCR7 combined with the catalytic triad might comprise the catalytic center. In CCR7, L192, F155, and H208 were identified as the substrate-binding sites, and site-directed mutations of these amino acids showed obvious changes in catalytic efficiency with respect to both feruloyl-CoA and sinapoyl-CoA. Mutant F155Y exhibited greater catalytic efficiency for sinapoyl-CoA compared with that of wild-type PtoCCR7. Finally, recent genome duplication events provided the foundation for CCR divergence. This study further identified the active sites in CCRs and the evolutionary process of CCRs in terrestrial plants.