Polyphosphate/ATP-dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK overexpression on lysine production.
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.
Lindner
Steffen N
SN
Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms University Muenster, 48149, Muenster, Germany.
Niederholtmeyer
Henrike
H
Schmitz
Katja
K
Schoberth
Siegfried M
SM
Wendisch
Volker F
VF
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008239:Lysine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011122:Polyphosphates / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011122:Polyphosphates; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; C019534:polyphosphate NAD-kinase; D008239:Lysine