In vitro ATP regeneration from polyphosphate and AMP by polyphosphate: AMPphosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthes is and biocatalysis. Several enzymatic ATP regeneration systems have been d escribed but have some disadvantages. We report here on the use of polyphos phate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) an d AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase a nd hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction po lyP(n) + AMP --> ADP + polyP(n-1). The ADP can be converted to ATP by adeny late kinase (AdK). Substrate specificity with nucleoside and 2'-deoxnucleos ide monophosphates was examined using partially purified PPT by measuring t he formation of nucleoside diphosphates with high-pressure liquid chromatog raphy. AMP and 2'-dAMP were efficiently phosphorylated to ADP and 2'-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the correspondin g diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP re generation using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl2, polyP(n=35), and AMP. Similar reaction mixtures containing strain 210A cell extract or part ially purified PPT, polyp, AMP, glucose, and hexokinase formed glucose 6-ph osphate. The results indicate that PPT from A. johnsonii is specific for AM P and 2'-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to exis ting enzymatic ATP regeneration systems in which phosphoenolpyruvate and ac etylphosphate serve as phosphoryl donors and has the advantage that AMP and polyp are stabile, inexpensive substrates.