Enzymatic Characteristics of a Polyphosphate/ATP-NAD Kinase, PanK, from Myxococcus xanthus.

Abstract

NAD kinase is a crucial enzyme for production of NADP+. Myxococcus xanthus is a gram-negative soil bacterium that forms fruiting bodies and spores under starvation, and it accumulates polyphosphate (poly(P)) during early development. We found that M. xanthus NAD kinase (PanK) utilized both ATP and poly(P) as phosphoryl donors; therefore, PanK was designated as a poly(P)/ATP-NAD kinase. Unlike other poly(P)/ATP-NAD kinases, PanK hardly exhibited NADH kinase activity. The NAD kinase activity of PanK was inhibited by NADPH, but not NADH. Replacement of Thr-90 in the GGDGT motif of PanK with Asn decreased both ATP- and poly(P)-dependent NAD kinase activities; however, poly(P)-dependent NAD kinase activity was further decreased by approximately 6- to 10-fold compared with ATP-dependent NAD kinase activity, suggesting that Thr-90 in the GGDGT motif of PanK may be important for poly(P) utilization. PanK preferred ATP and short-chain poly(P) as phosphoryl donors. The Km of PanK for ATP, poly(P)4, and poly(P)10-15 was 0.66mM, 0.08mM, and 0.71mM, respectively, and the catalytic efficiency (kcat/Km) for poly(P)4 was 2.4-fold higher than that for ATP, suggesting that M. xanthus under starvation conditions may be able to efficiently generate NADP+ using PanK, ATP, and poly(P).