Systematic characterization of the ADP-ribose pyrophosphatase family in the cyanobacterium Synechocystis sp strain PCC 6803
Systematic characterization of the ADP-ribose pyrophosphatase family in the cyanobacterium Synechocystis sp strain PCC 6803
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DOI：
10.1128/jb.187.14.4984-4991.2005
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发表时间：
2005-07-01
影响因子：
3.2
通讯作者：
Nishiyama, Y
中科院分区：
文献类型：
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作者：
Okuda, K;Hayashi, H;Nishiyama, Y
我们在蓝藻集胞藻属中表征了四种假定的 ADP-核糖焦磷酸酶 SII1054、SIr0920、SIr1134 和 SIr1690。菌株PCC 6803。每种重组蛋白在大肠杆菌中过表达并纯化。 SII1054和Sh-0920特异性水解ADP-核糖，而SIr1134不仅水解ADP-核糖，还水解NADH和黄素腺嘌呤二核苷酸。相比之下，Slr1690 对 ADP-核糖表现出非常低的活性，并且在 Nudix 基序中具有四个氨基酸取代，表明 Slr1690 不是活性 ADP-核糖焦磷酸酶。然而，Slr1690 T73G/I88E/K92E/A94G 的四重突变用 Nudix 基序中的保守氨基酸替换了突变的氨基酸，导致 k(cat) 值显着增加（6.1 X 10(2) 倍）。这些结果表明 Slr1690 可能是从活性 ADP-核糖焦磷酸酶进化而来的。功能和聚类分析表明，SII1054 是一种细菌类型，而其他三种和 SIr0787（之前已进行过表征（Raffaelli 等人，FEBS Lett. 444:222-226, 1999））是系统发育上不同的类型，通过分子进化机制（例如结构域融合和氨基酸取代）起源于古细菌 Nudix 蛋白。
We have characterized four putative ADP-ribose pyrophosphatases SII1054, SIr0920, SIr1134, and SIr1690 in the cyanobacterium Synechocystis sp. strain PCC 6803. Each of the recombinant proteins was overexpressed in Escherichia coli and purified. SII1054 and Sh-0920 hydrolyzed ADP-ribose specifically, while SIr1134 hydrolyzed not only ADP-ribose but also NADH and flavin adenine dinucleotide. By contrast, Slr1690 showed very low activity for ADP-ribose and had four substitutions of amino acids in the Nudix motif, indicating that Slr1690 is not an active ADP-ribose pyrophosphatase. However, the quadruple mutation of Slr1690, T73G/ I88E/K92E/A94G, which replaced the mutated amino acids with those conserved in the Nudix motif, resulted in a significant (6.1 X 10(2)-fold) increase in the k(cat) value. These results suggest that Slr1690 might have evolved from an active ADP-ribose pyrophosphatase. Functional and clustering analyses suggested that SII1054 is a bacterial type, while the other three and SIr0787, which was characterized previously (Raffaelli et al., FEBS Lett. 444:222-226, 1999), are phylogenetically diverse types that originated from an archaeal Nudix protein via molecular evolutionary mechanisms, such as domain fusion and amino acid substitution.