A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism
A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism
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DOI：
10.1042/bj20080714
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发表时间：
2008-09-01
影响因子：
4.1
通讯作者：
Grogan, Gideon
中科院分区：
文献类型：
--
作者：
Bennett, Joseph P.;Bertin, Lucille;Grogan, Gideon
羟基肉桂酰辅酶A水合酶(HCHL)催化阿魏酰辅酶A生物转化为乙酰辅酶A和重要的香草醛(4-羟基-3-甲氧基苯甲醛)，用于阿魏酸生物转化为天然香草醛。在HCHL的催化下，反应分为两步进行，首先阿魏酰辅酶A的双键水合，然后通过逆醛缩合反应裂解生成的β-羟基硫酸酯，生成产物。利用HCHL的晶体结构对活性中心残基进行的动力学分析表明，虽然Glu-143是活性所必需的，但Ser-123在催化中没有主要作用。但是，Tyr-239突变为Phe后，底物结合的K-M值大大增加。WT(野生型)HCHL和S123A突变体的结构都与阿魏酰辅酶A共结晶，在配体结合时显示出微妙的螺旋运动，其结果是使Tyr-239的酚羟基从相邻的亚基接近Tyr-75，以便与产物香兰素的酚羟基结合，观察到其电子密度。与配体结合的HCHL的活性中心残基与结构无关的香草醇氧化酶的活性中心残基显示出显著的三维重叠，香草醇氧化酶也识别与香草醛相关的对羟基芳香底物。这些数据既解释了HCHL对对羟基肉桂酸衍生物的底物专一性，也说明了这两种原本不相关的酶之间配体识别的分子决定因素的显着趋同。
HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxy-benzaldehyde) and is exploited in whole-cell systems for the bio-conversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K-M for the substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.