Identification and characterization of NuhA, a novel Nudix hydrolase specific for ADP-ribose in the cyanobacterium Synechococcus sp PCC 7002
Identification and characterization of NuhA, a novel Nudix hydrolase specific for ADP-ribose in the cyanobacterium Synechococcus sp PCC 7002
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DOI：
10.1016/j.bbapap.2004.03.004
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发表时间：
2004-06-01
影响因子：
3.2
通讯作者：
Hayashi, H
中科院分区：
文献类型：
--
作者：
Okuda, K;Nishiyama, Y;Hayashi, H
我们克隆了蓝细菌聚球藻属PCC 7002中一种新的营养素水解酶的基因，并将其命名为nuhA。推导的氨基酸序列包括NuhA基序GX（5）EX（7）EXEEXGV，这是NuhA水解酶共有的基序，此外，NuhA基序C-末端第15个氨基酸处的脯氨酸，这是ADP-核糖焦磷酸酶亚家族的特征。在大肠杆菌中过表达并纯化具有六组氨酸标签的重组NuhA。重组NuhA在各种核苷二磷酸衍生物中特异性地水解ADP-核糖。水解ADP-核糖的活性需要Mg~（2+），最适pH为9.5。水解的V-max和K-m值分别为23.6单位mg（-1）和0.094 mM。NuhA在C-末端区域含有一个未表征的结构域，称为Pfam-B-3116，其在几种假设蛋白中是保守的。Pfam-B-3116结构域缺失的突变NuhA不能形成NuhA特有的六聚体，并且对ADP-核糖表现出显著更高的Km值，这表明Pfam-B-3116结构域可能负责NuhA的寡聚化和对ADP-核糖的完全结合亲和力。这些独特的功能表明，NuhA是一种新型的ADP-核糖焦磷酸酶。(C)2004 Elsevier B.V.保留所有权利。
We cloned the gene for a novel Nudix hydrolase in the cyanobacterium Synechococcus sp. PCC 7002 and termed it nuhA. The deduced amino acid sequence of NuhA included the Nudix motif, GX(5)EX(7)RELXEEXGV, which is common to Nudix hydrolases, and in addition, a proline at the 15th amino acid from the C-terminus of the Nudix motif, which is characteristic of the subfamily of ADP-ribose pyrophosphatases. The recombinant NuhA with a hexahistidine tag was overexpressed in Escherichia coli and purified. The recombinant NuhA hydrolyzed ADP-ribose specifically among various nucleoside diphosphate derivatives. The hydrolytic activity for ADP-ribose required Mg2+ and was optimal at pH 9.5. The V-max and K-m values of hydrolysis were 23.6 units mg(-1) and 0.094 mM, respectively. NuhA contained an uncharacterized domain in the C-terminal region, termed Pfam-B-3116, which is conserved in several hypothetical proteins. The mutated NuhA deficient in the Pfam-B-3116 domain failed to form the hexamers that are characteristic of NuhA, and exhibited a significantly higher Km value for ADP-ribose, suggesting that the Pfam-B-3116 domain might be responsible for oligomerization of NuhA and full binding affinity for ADP-ribose. These unique features suggest that NuhA is a novel type of ADP-ribose pyrophosphatase. (C) 2004 Elsevier B.V. All rights reserved.