His-234是毛白杨对香豆酸:CoA连接酶的重要酶催化活性残基  

His-234 is an Essential Residue for Populus tomentosa 4-coumarate: CoA Ligase Enzymatic Mechanisms

作　　者：任百光 李德峰[2] 郑彩霞[1] 盖颖[1] 蒋湘宁[1] 胡永林[2] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083 [2]中国科学院生物物理研究所生物大分子国家重点实验室,北京100101

出　　处：《生物化学与生物物理进展》2013年第11期1165-1172,共8页Progress In Biochemistry and Biophysics

基　　金：国家高技术研究发展计划(863)(2011AA100203);国家重点基础研究发展计划(973)(2011CB910300;2011CB911100)资助项目~~

摘　　要：对香豆酸∶CoA连接酶(4-coumarate:coenzyme A ligase,4CL)是植物苯丙烷类代谢途径中的一个重要的酶.4CL以肉桂酸衍生物(香豆酸、咖啡酸、阿魏酸等)、ATP和CoA为底物合成相应的酰基-CoA酯,这些酰基-CoA酯是一系列重要化合物(如木质素)的前体.4CL的酶催化反应分两步进行:第一步以肉桂酸衍生物和Mg2+-ATP为底物合成酰基-AMP,第二步用CoA取代AMP,产生酰基-CoA酯,催化过程中酶的构象产生明显的变化.因为4CL在木质素的合成中所起的作用,这个酶是通过蛋白质工程方法改进林产品质量的重要靶标.我们通过X射线衍射技术,解析了毛白杨对香豆酸∶CoA连接酶1(Pt4CL1)与其中间产物对香豆酰-AMP的复合物晶体结构,与同家族成员结构比对,确定所获得的蛋白质结构为Pt4CL1催化第二步反应,即酰基-CoA酯合成的构象.结构分析表明:His-234残基在Pt4CL1的酶催化机理中起着多重作用,即通过侧链与AMP磷酸基团形成氢键,降低磷酸基团的负电荷,催化CoA的亲核取代反应;侧链可以采取两种不同的构象以调节CoA进入Pt4CL1的催化中心;His-234的侧链还可能夺取CoA巯基的质子,从而增强CoA的亲核反应活性.突变体酶活数据结果也显示His-234对Pt4CL1的活性非常重要,是Pt4CL1催化中心的活性残基.The 4-coumarate ： CoA ligase （4CL） is one of the most important enzymes of the plant specific phenylpropanoid pathway. It catalyzes the syntheses of cournaroyl-CoA thioesters, the precursors of lignin and other important phenylpropanoids, from corresponding coumarate compounds such as 4-coumaric acid, caffeic acid, and 3-methoxy-4-coumaric acid, ATP, and coenzyme A, in two-step reactions that involves the formation of coumarate-AMP anhydride as intermediates and subsequent formation of the the coumaroyl-CoA thioesters through the nucleophilic substitution of the AMP group by CoA. Because of the important roles played by 4CL in the biosynthesis of lignin, this protein has become the target of bioengineering aimed at improving the quality of plant products. We have solved the crystal structure of Populus tomentosa 4CL1 （Pt4CL1） complexed with its enzymatic intermediate 4-coumarate-AMP anhydride. The Pt4CL1 was found to adopt the conformation of the second step reaction based on comparisons with other members from the same superfamily. Structural analysis revealed that the residue His-234, which forms a hydrogen bond to the phosphate group of the 4-coumarate-AMP anhydride through its side chain imidazole group, plays essential roles in the enzymatic mechanisms of Pt4CL1, including facilitating both steps of the reactions by forming a hydrogen bond to, and reducing the negative charge of, the phosphate group; de-protonating the thiol group of CoA; and regulating the access of the active site to CoA through the conformational changes of its side chain. Enzymatic assays on Pt4CL1 and relevant mutant verified that His-234 is an essential residue of this enzyme.

关 键 词：木质素合成 4CL 酶活机理 晶体结构 结构-功能关系 

分 类 号：Q71[生物学—分子生物学]