Chemical mechanism and substrate specificity of RhlI, an acylhomoserine lactone synthase from Pseudomonas aeruginosa
Chemical mechanism and substrate specificity of RhlI, an acylhomoserine lactone synthase from Pseudomonas aeruginosa
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DOI：
10.1021/bi048005m
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发表时间：
2005-03-01
期刊：
影响因子：
2.9
通讯作者：
Tipton, PA
中科院分区：
文献类型：
--
作者：
Raychaudhuri, A;Jerga, A;Tipton, PA
RhlI酶催化s -腺苷蛋氨酸和n -丁基酰基载体蛋白形成n -丁基高丝氨酸内酯。n -丁基同丝氨酸内酯在铜绿假单胞菌中作为群体感应信号分子，参与调控细菌毒力和感染性的许多过程。铜绿假单胞菌基因组包含三个编码酰基载体蛋白的基因。我们克隆了这三个基因，表达了酰基载体蛋白，并将它们描述为RhlI的底物。建立了一种连续分光光度测定法，以促进RhlI的动力学和机理研究。Acp1是一个较好的RhlI底物，其K-m为7 muM；反应进行时k（cat）值为0.35 s（-1）。支持脂肪酸生物合成的AcpP也是RhlI反应的良好底物，k（cat）为0.46 s(-1)， AcpP的k -m为6 muM。第三个酰基载体蛋白Acp3是一个较差的RhlI底物，K-m为280 muM；K （cat）为0.03 s（-1）。结合文献中显示编码Acp1基因的基因表达受群体感应系统控制的微阵列数据，我们的数据表明Acp1可能是体内RhlI的底物。同位素标记研究了rhli催化内酯化反应的化学机理。溶剂的氘核没有被纳入产物，这暗示了一种直接的攻击机制，即假定的n -丁基- sam中间体的羧酸氧攻击与磺酸离子相邻的亚甲基碳。通过消除甲基硫腺苷形成n -丁基乙烯基甘氨酸的其他机制被排除，基于RhlI未能将真正的n -丁基乙烯基甘氨酸转化为n -丁基- l-同丝氨酸内酯的观察。
The enzyme RhlI catalyzes the formation of N-butyrylhomoserine lactone from S-adenosylmethionine and N-butyrylacyl carrier protein. N-Butyrylhomoserine lactone serves as a quorum-sensing signal molecule in Pseudomonas aeruginosa, and is implicated in the regulation of many processes involved in bacterial virulence and infectivity. The P. aeruginosa genome contains three genes encoding acyl carrier proteins. We have cloned all three genes, expressed the acyl carrier proteins, and characterized each as a substrate for RhlI. A continuous, spectrophotometric assay was developed to facilitate kinetic and mechanistic studies of RhlI. Acp1, which has not been characterized previously, was a good substrate for RhlI, with a K-m of 7 muM; the reaction proceeded with a k(cat) value of 0.35 s(-1). AcpP, which supports fatty acid biosynthesis, was also a good substrate in the RhlI reaction, where k(cat) was 0.46 s(-1), and the K-m for AcpP was 6 muM. The third acyl carrier protein, Acp3, was a poor substrate for RhlI, with a K-m of 280 muM; k(cat) was 0.03 s(-1). Taken together with microarray data from the literature which show that expression of the gene encoding Acp1 is under the control of the quorum-sensing, system, our data suggest that Acp1 is likely to be the substrate for RhlI in vivo. Isotope labeling studies were conducted to investigate the chemical mechanism of the RhlI-catalyzed lactonization reaction. Solvent deuterons were not incorporated into product, which implicates a direct attack mechanism in which the carboxylate oxygen of the presumptive N-butyryl-SAM intermediate attacks the methylene carbon adjacent to the sulfonium ion. Alternative mechanisms, in which N-butyrylvinylglycine is formed via elimination of methylthioadenosine, were ruled out on the basis of the observation that RhlI failed to convert authentic N-butyrylvinylglycine to N-butyryl-L-homoserine lactone.