Two isocitrate dehydrogenases from a plant pathogen Xanthomonas campestris pv. campestris 8004. Bioinformatic analysis, enzymatic characterization, and implication in virulence
Two isocitrate dehydrogenases from a plant pathogen Xanthomonas campestris pv. campestris 8004. Bioinformatic analysis, enzymatic characterization, and implication in virulence
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来自植物病原体野油菜黄单胞菌 pv. 的两种异柠檬酸脱氢酶。
DOI：
10.1002/jobm.201500648
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发表时间：
2016-09-01
影响因子：
3.1
通讯作者：
Zhu, Guoping
中科院分区：
文献类型：
--
作者：
Lv, Changqi;Wang, Peng;Zhu, Guoping
异柠檬酸脱氢酶（IDH）是三羧酸循环（TCA）中的关键酶，可能在致病菌的毒力中起重要作用。在这里，两个结构不同的IDH从植物病原体野油菜黄单胞菌致病变种。campestris 8004（XccIDH1和XccIDH2）的基因型进行了详细的鉴定。重组XccIDH1在溶液中形成同源二聚体，而重组XccIDH2是典型的单体。系统发育分析表明，XccIDH1属于IDH亚家族I型，XccIDH2属于IDH单体进化枝。动力学特性表明，XccIDH1对NAD（+）的特异性是NADP（+）的110倍，而XccIDH2对NADP（+）的特异性是NAD（+）的353倍。通过定点突变研究了推定的辅酶鉴别氨基酸（对于XccIDH1为Asp268、Ile269和Ala275，对于XccIDH2为Lys589、His590和Arg601）。两个突变体mXccIDH1和mXccIDH2的辅酶特异性分别从NAD（+）完全逆转为NADP（+），从NADP（+）完全逆转为NAD（+）。此外，XccIDH1的Ser80和XccIDH2的Lys256和Tyr421是底物结合的决定因素。进一步研究了XccIDH1和XccIDH2的最适pH、最适温度、热稳定性和金属离子效应等生化特性。本文还对这两种异黄酮类化合物作为防治Xcc 8004引起的植物病害的药物开发靶点的可能性进行了深入的讨论。
Isocitrate dehydrogenase (IDH) is a key enzyme in the tricarboxylate (TCA) cycle, which may play an important role in the virulence of pathogenic bacteria. Here, two structurally different IDHs from a plant pathogen Xanthomonas campestris pv. campestris 8004 (XccIDH1 and XccIDH2) were characterized in detail. The recombinant XccIDH1 forms homodimer in solution, while the recombinant XccIDH2 is a typical monomer. Phylogenetic analysis showed that XccIDH1 belongs to the type I IDH subfamily and XccIDH2 groups into the monomeric IDH clade. Kinetic characterization demonstrated that XccIDH1's specificity towards NAD(+) was 110-fold greater than NADP(+), while XccIDH2's specificity towards NADP(+) was 353-fold greater than NAD(+). The putative coenzyme discriminating amino acids (Asp268, Ile269 and Ala275 for XccIDH1, and Lys589, His590 and Arg601 for XccIDH2) were studied by site-directed mutagenesis. The coenzyme specificities of the two mutants, mXccIDH1 and mXccIDH2, were completely reversed from NAD(+) to NADP(+), and NADP(+) to NAD(+), respectively. Furthermore, Ser80 of XccIDH1, and Lys256 and Tyr421 of XccIDH2, were the determinants for the substrate binding. The detailed biochemical properties, such as optimal pH and temperature, thermostability, and metal ion effects, of XccIDH1 and XccIDH2 were further investigated. The possibility of taking the two IDHs into consideration as the targets for drug development to control the plant diseases caused by Xcc 8004 were described and discussed thoroughly.