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首页> 外文期刊>Journal of Molecular Biology >Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis
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Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis

机译：结核分枝杆菌Rv0805环状核苷酸磷酸二酯酶的结构和生化分析

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摘要

Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We is have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, an unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe 3,-Mn2+ binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn2+ co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class Ill PDEs.
机译：细菌和真核生物中环核苷酸单磷酸酯（cNMP）的水解是由不同的cNMP磷酸二酯酶（PDE）引起的。由于这些酶的氨基酸序列和性质不同，因此它们通过融合进化而进化。环状NMP PDE将cNMP裂解为NMP，而Rv0805基因产物是迄今为止在结核分枝杆菌基因组中唯一可识别的cNMP PDE。我们已经显示，Rv0805是cAMP / cGMP双重特异性PDE，其氨基酸序列与哺乳动物cNMP PDE无关。 Rv0805是二聚体Fe 3，-Mn2 +双核PDE，突变分析表明，活性位点金属由保守的天冬氨酸，组氨酸和天冬酰胺残基配位。我们在这里报告Rv0805的催化核心的结构，它与钙调磷酸酶样磷酸酶有很远的关系。 Rv0805二聚体的晶体结构表明，活性位点金属有助于二聚作用，因此除参与催化作用外，还发挥了其他结构作用。我们还介绍了缺少Mn2 +配位残基之一的Asn97Ala突变蛋白的晶体结构，以及具有受损的cAMP水解活性的Asp66Ala突变体，为这些突变蛋白的催化特性提供了结构基础。磷酸分子以二齿方式结合在Rv0805野生型蛋白的活性位点，而甲草胺在Asp66Ala突变蛋白的晶体结构中占据相似的位置。通过计算对接研究鉴定了Rv0805中独特的底物结合口袋，并通过突变分析验证了His140残基在与cAMP相互作用中的作用。因此，有关细菌cNMP PDE的第一个结构的报告显着扩展了我们对Ill PDE类中cAMP水解的分子理解。

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