Figure 3.

Enzymatic pathways to make nucleoside triphosphates (right, magenta) from nucleosides (left, blue). The standard pathway concept (center) adds single phosphate groups stepwise, one at a time, to convert a nucleoside to its nucleoside monophosphate, the nucleoside monophosphate to its nucleoside diphosphate, and the nucleoside diphosphate to its nucleoside triphosphate, with ATP providing the phosphate moiety at each step. It has proven difficult to find a natural nucleoside monophosphate kinase, or to engineer one, to accept triphosphates that have non-standard nucleobases (Figs. 1 and 2). Accordingly, polyphosphate kinases (green) were sought to convert monophosphates to di- and triphosphates using polyphosphate as a phosphate donor.