Overexpression, purification, and characterization of ATP-NAD kinase of Sphingomonas sp A1
Overexpression, purification, and characterization of ATP-NAD kinase of Sphingomonas sp A1
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DOI：
10.1016/j.pep.2004.03.012
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发表时间：
2004-07-01
影响因子：
1.6
通讯作者：
Murata, K
中科院分区：
文献类型：
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作者：
Ochiai, A;Mori, S;Murata, K
神经鞘氨醇单胞菌NAD激酶基因(NadK)。将A1基因克隆并在大肠杆菌中高效表达，通过5步纯化，获得了活性为25%的NadK蛋白。NADK是一个32 kDa亚基的同源二聚体，利用三磷酸腺苷或其他核苷三磷酸盐，而不是无机多聚磷酸盐，作为磷酸供体来催化NAD的磷酸化，在pH 8.0和50-55℃时最有效，被命名为ATP-NAD激酶(NADK)。NADK无NADH激酶活性，NADP(H)对其有轻度抑制作用。NAD生物合成的前体如喹啉酸、烟酸单核苷酸、烟酸腺嘌呤二核苷酸和烟酸对NADK活性没有影响，就像在黄色微球菌、结核分枝杆菌和大肠杆菌的NAD酶的情况下观察到的那样。连同枯草芽孢杆菌的NAD激酶被喹啉酸激活的报道[J.细菌素。185(2003)4844]，研究表明，即使在细菌的NAD激酶中，其调节模式也是不同的。(C)2004 Elsevier Inc.保留所有权利。
The NAD kinase gene (nadK) of Sphingomonas sp. A1 was cloned and then overexpressed in Escherichia coli, and the gene product (NadK) was purified from the E coli cells through five steps with a 25% yield of activity. NadK was a homodimer of 32 kDa subunits, utilized ATP or other nucleoside triphosphates, but not inorganic polyphosphates, as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 8.0 and 50-55 degreesC, and was designated as ATP-NAD kinase (NadK). NadK showed no NADH kinase activity and was slightly inhibited by NADP(H). Precursors for NAD biosynthesis such as quinolinic acid, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, and nicotinic acid had no effect on the NadK activity, as observed in the cases of the NAD kinases of Micrococcus flavus, Mycobacterium tuberculosis, and E coli. Taken together with the report that the NAD kinase of Bacillus subtilis is activated by quinolinic acid [J. Bacteriol. 185 (2003) 4844], it is indicated that the regulatory patterns of NAD kinases differ even among bacterial NAD kinases. (C) 2004 Elsevier Inc. All rights reserved.