Molecular characterization of Escherichia coli NAD kinase
Molecular characterization of Escherichia coli NAD kinase
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DOI：
10.1046/j.1432-1327.2001.02358.x
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发表时间：
2001-08-01
期刊：
影响因子：
--
通讯作者：
Murata, K
中科院分区：
文献类型：
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作者：
Kawai, S;Mori, S;Murata, K
从大肠杆菌MG 1655中纯化NAD激酶至均一。该酶是由30 kDa亚基组成的六聚体，并利用ATP或其他核苷三磷酸作为磷酸化NAD的磷酰基供体，在pH 7.5和60 ℃下最有效。该酶不能利用无机多磷酸作为磷酰供体，被命名为ATP-NAD激酶。该酶的N端氨基酸序列由yfjB编码，该基因已作为一个功能未知的基因在大肠杆菌中保藏。coli全基因组DNA序列数据库yfjB的克隆和表达。coli BL21（DE3）pLysS.纯化产物（YfjB）具有NAD激酶活性，与E. coli MG 1655的分子结构和其他酶学性质。推导的YfjB的氨基酸序列与结核分枝杆菌无机多磷酸盐/ATP-NAD激酶的氨基酸序列具有同源性[Kawai，S.，Mori，S.，穆凯，T.，铃木，S.，桥本，W.，Takeshi，Y. & Murata，K.等人（2000）Biochem. Biophys.通信资源276，57-63]，以及那些功能尚未被揭示的许多假设蛋白质。YfjB同源物被认为是NAD激酶，并且它们的序列比对显示高度保守的区域XXX-XGGDG-XL和DGXXX-TPTGSTAY，其中X代表疏水氨基酸残基。
NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 degreesC. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP-NAD kinase. The N-terminal amino-acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence databases yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP-NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. The deduced amino-acid sequence of YfjB exhibited homology with that of Mycobacterium tuberculosis inorganic polyphosphate/ATP-NAD kinase [Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto, W., Takeshi, Y. & Murata, K. (2000) Biochem. Biophys. Res. Commun. 276, 57-63], and those of many hypothetical proteins for which functions have not yet been revealed. The YfjB homologues were considered to be NAD kinases and alignment of their sequences revealed highly conserved regions, XXX-XGGDG-XL and DGXXX-TPTGSTAY, where X represents a hydrophobic amino-acid residue.