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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.
4-coumarate: coenzyme A ligase (4CL) is an important enzyme in plant phenylpropanoid metabolic pathway.4CL is synthesized with cinnamic acid derivatives (coumaric acid, caffeic acid, ferulic acid Etc.), ATP and CoA as substrates to synthesize the corresponding acyl-CoA esters, which are precursors of a series of important compounds such as lignin.The enzymatic catalysis of 4CL proceeds in two steps: Cinnamic acid derivatives and Mg2 + -ATP substrate for the synthesis of acyl-AMP, the second step with CoA instead of AMP to produce acyl-CoA ester, catalytic conformation of the enzyme produced significant changes because 4CL in the synthesis of lignin This enzyme is an important target for improving the quality of forest products through protein engineering methods.We analyzed the effects of coumaric acid-CoA ligase 1 (Pt4CL1) and its intermediate on coumaroyl- AMP crystal structure of the complex with the same family member structure to determine the structure of the obtained protein Pt4CL1 catalyzed the second reaction that the acyl-CoA ester synthesis conformation. Structural analysis showed that: His-234 residues in Pt4CL1 Enzymatic catalysis plays a multiple role, through side chains and AMP Acid groups form hydrogen bonds, reducing the negative charge of the phosphate group and catalyzing the nucleophilic substitution reaction of CoA. Two different conformations of the side chain can take place to regulate the CoA into the catalytic center of Pt4CL1. The side chain of His-234 may also take CoA thiol proton to enhance the nucleophilic reactivity of CoA.Results of mutant enzyme activity also showed that His-234 is very important for the activity of Pt4CL1 and is the active residue of Pt4CL1 catalytic center.