The mechanism underlying the acquisition of specificity for ATP has been elucidated by researchers in a new report published in Scientific Reports on September 11th, 2013.
Inorganic polyphosphate [poly(P)], a polymer of orthophosphate (Pi) residues linked by high-energy phosphoanhydride bonds, is found in every organism from bacteria to animals (Fig.1). ATP is a universal energy carrier, sometimes termed the "energy currency" of cells (Fig.1). NAD kinase (NADK) catalyzes the phosphorylation of NAD+ to yield NADP+, using poly(P) or ATP as a source of phosphate and energy. poly(P)/ATP-NADK utilizes both poly(P) and ATP, whereas ATP-specific NADK prefers ATP to poly(P).
The group have assumed that NADKs evolved from poly(P)/ATP-NADKs into ATP-specific NADKs, and that recently diverged organisms obtained ATP-specific NADKs during their evolution, based on the following observations: (i) it has been deduced that bacteria evolved from Gram-positive bacteria or Archaea into Gram-negative ?-proteobacteria; (ii) poly(P)/ATP-NADKs are distributed throughout Gram-positive bacteria and Archaea, whereas ATP-specific NADKs are found in Gram-negative ?-proteobacteria (Escherichia coli and Salmonella enterica) and eukaryotes. However, it remains unclear how NADKs evolved from poly(P)/ATP-NADKs into ATP-specific NADKs.
In this study, we succeeded in conferring the ability to utilize poly(P) on ?-proteobacterial ATP-specific NADKs, and hence in the effective "reversion" of these NADKs, through a single amino-acid substitution: substitution of Asn to Thr in the GGDGN motif that is conserved in the ?-proteobacterial ATP-specific NADKs. As a result of this substitution, the poly(P)-dependent NADK activity of E. coli ATP-specific NADK dramatically increased, from 2.8? to 43? (the relative activity to the 100? ATP-dependent activity). These data indicate that NADK has lost the ability to utilize poly(P), and that it acquired its specificity for ATP over the course of evolution as a result of the change from Thr to Asn in the GGDGT motif conserved among NADKs from almost all organisms.
Remarkably, almost all ?-proteobacterial NADKs carrying the GGDGN motif are further concentrated within the most recently diverged group within ?-proteobacteria, which includes E. coli and other pathogenic bacteria (Salmonella, Shigella, Yersinia, Vibrio, etc.) (Fig.2). Therefore, we propose that NADKs evolved from poly(P)/ATP-NADKs (carrying GGDGT) via Gram-negative ?-proteobacterial poly(P)/ATP-NADKs (carrying GGDGS) into Gram-negative ?-proteobacterial ATP-specific NADKs (carrying GGDGN) (Fig.2), as also depicted in Fig.3. It should be noted that human NADK carries the GGDGT motif, although this enzyme is ATP-specific, indicating that the mechanism underlying the acquisition of specificity for ATP (i.e., the structural determinants conferring specificity for ATP) differs between human ATP-specific NADK and ?-proteobacterial ATP-specific NADKs.
The group have established methods for large-scale production of NADP+, which is used in the diagnostic pharmaceutical industry, by utilizing poly(P)/ATP-NADK (Patent: 4088251 JAPAN). However, we have shown that the poly(P)/ATP-NADK created through the method described here exhibits higher poly(P)-dependent activity, and is therefore more suitable for poly(P)-dependent mass production of NADP+, than the poly(P)/ATP-NADK the group used originally.
Because the structural determinants conferring specificity to ATP differ between human ATP-specific NADK and ?-proteobacterial ATP-specific NADKs, it might be possible to develop new antibiotics that specifically target ?-proteobacterial ATP-specific NADKs, but not human ATP-specific NADK. This study also provides a significant clue regarding how NADKs recognize and utilize poly(P). This information opens the way to development of a novel system for production of valuable compounds, in which the ability to utilize poly(P) would be conferred on various ATP-specific enzymes, and the resultant poly(P)-dependent enzymes could then be utilized to produce various phosphoryl compounds using poly(P) instead of ATP. Finally, this study provides significant hints regarding why poly(P)/ATP-NADK has evolved into ATP-specific NADK, what the physiological significance of this evolution is, and what the relationship of this evolution with the properties (pathogenicity, etc.) of each bacterium is.
Yusuke, N. et al. Conferring the ability to utilize inorganic polyphosphate on ATP-specific NAD kinase, Scientific Reports 3, 2632. DOI: 10.1038/srep02632 (2013)