Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms University Muenster, 48149, Muenster, Germany.
Appl Microbiol Biotechnol. 2010 Jun;87(2):583-93. doi: 10.1007/s00253-010-2481-y. Epub 2010 Feb 24.
Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 +/- 0.006 mol lysine (mol glucose)(-1), which was 12% higher than that of the empty vector control strain.
烟酰胺腺嘌呤二核苷酸磷酸（NADP）是通过氧化或还原的烟酰胺腺嘌呤二核苷酸（NAD/NADH）的磷酸化合成的。在这里，从谷氨酸棒杆菌基因组中纯化了 cg1601/ppnK 基因产物，从重组大肠杆菌中进行酶学表征，揭示了其作为多磷酸盐（PolyP）/ATP 依赖性烟酰胺激酶（PPNK）的活性。来自谷氨酸棒杆菌的 PPNK 作为同源四聚体是活跃的，可接受 PolyP、ATP，甚至 ADP 用于 NAD 的磷酸化。用 ATP 作为磷酸供体进行 NAD 磷酸化的催化效率高于 PolyP。就 PolyP 的链长而言，PPNK 可与短链 PolyP 一起发挥作用。当使用 ATP 时，PPNK 活性不依赖于二价阳离子，但被锰离子增强，特别是镁离子。当使用 PolyP 时，PPNK 需要二价阳离子，优选锰离子，才能发挥活性。PPNK 被 NADP 和 NADH 抑制，其浓度低于毫摩尔。在谷氨酸棒杆菌野生型中过表达 ppnK 会轻微降低生长速度，而在赖氨酸生产菌株 DM1729 中过表达 ppnK 会导致葡萄糖上赖氨酸产物的产率为 0.136 +/- 0.006 mol 赖氨酸（mol 葡萄糖）（-1），比空载体对照菌株高 12%。
