Overexpression, purification, and characterization of ATP-NAD kinase of Sphingomonas sp. A1.
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 degrees C, 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.
Ochiai
Akihito
A
Department of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, 611-0011, Japan.
Mori
Shigetarou
S
Kawai
Shigeyuki
S
Murata
Kousaku
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D009249:NADP / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics
D002413:Cations, Divalent; D009249:NADP; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase