Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism
Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism
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DOI：
10.1074/jbc.m209362200
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发表时间：
2003-01-03
影响因子：
4.8
通讯作者：
Legrand, M
中科院分区：
文献类型：
--
作者：
Hoffmann, L;Maury, S;Legrand, M
通过一系列高压液相色谱步骤，从烟草茎提取物中纯化了一种水解羟基肉桂酰辅酶A酯的蛋白质。其N-末端氨基酸序列的测定允许设计引物，允许通过PCR克隆相应的cDNA。序列分析表明，该基因属于植物酰基转移酶基因家族，其成员具有多种功能。烟草cDNA在细菌细胞中表达为与谷胱甘肽S-转移酶融合的重组蛋白。融合蛋白经亲和纯化和切割，得到重组酶，用于催化性质的研究。该酶催化合成的莽草酸和奎尼酸酯最近被证明是参与苯丙素类生物合成的细胞色素P450 3-羟化酶的底物。这种酶被命名为羟基肉桂酰辅酶A：莽草酸/奎尼酸羟基肉桂酰转移酶。我们发现，p-香豆酰辅酶A和咖啡酰辅酶A是最好的酰基基团的捐助者和酰基基团更有效地转移到莽草酸比奎尼酸。该酶还催化逆反应，即从绿原酸（5-O-咖啡酰奎尼酸酯）形成咖啡酰辅酶A。因此，羟基肉桂酰-CoA：莽草酸/奎尼酸羟基肉桂酰转移酶似乎控制主要植物酚类化合物如木质素和绿原酸的生物合成和周转。
A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/ quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.