In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

Abstract

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP n ؉ AMP 3 ADP ؉ polyP n؊1 . The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2-dAMP were efficiently phosphorylated to ADP and 2-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration 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, MgCl 2 , polyP n‫53؍‬ , and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP 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 existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.Enzyme-catalyzed phosphoryl transfer reactions (i.e., those which form or cleave P-O bonds) represent a viable alternative to multistep chemical phosphorylations in preparative organic synthesis (7,10,20). Many useful phosphorylating enzymes require nucleoside triphosphates as cofactors. ATP is the most important biological phosphate donor and is a required cofactor for numerous enzymatic reactions in both anabolic and catabolic metabolism, specifically in the formation of P-O bonds. Cofactor provision for ATP-dependent enzymes can be accomplished by their direct addition in stoichiometric amounts or by the inclusion of a cofactor regeneration system. Direct cofactor addition is not only costly but can unfavorably alter reaction equilibrium, lead to an accumulation of inhibitory cofactor by-products, and complicate recovery of end products (20). For preparative-scale organic synthesis, these problems have been overcome by the development of enzymatic systems for ATP regeneration.Whitesi...

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