Conferring the ability to utilize inorganic polyphosphate on ATP-specific NAD kinase

Yusuke Nakamichi, Aya Yoshioka, Shigeyuki Kawai, Kousaku Murata

NAD kinase (NADK) is a crucial enzyme for production of NADP⁺. ATP-specific NADK prefers ATP to inorganic polyphosphate [poly(P)] as a phosphoryl donor, whereas poly(P)/ATP-NADK utilizes both ATP and poly(P), and is employed in industrial mass production of NADP⁺. Poly(P)/ATP-NADKs are distributed throughout Gram-positive bacteria and Archaea, whereas ATP-specific NADKs are found in Gram-negative α- and γ-proteobacteria and eukaryotes. In this study, we succeeded in conferring the ability to utilize poly(P) on γ-proteobacterial ATP-specific NADKs through a single amino-acid substitution; the substituted amino-acid residue is therefore important in determining the phosphoryl-donor specificity of γ-proteobacterial NADKs. We also demonstrate that a poly(P)/ATP-NADK created through this method is suitable for the poly(P)-dependent mass production of NADP⁺. Moreover, based on our results, we provide insight into the evolution of bacterial NADKs, in particular, how NADKs evolved from poly(P)/ATP-NADKs into ATP-specific NADKs.