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 --> 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(n=35), 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.

FIG. 1

General scheme showing enzymatic ATP regeneration from AMP and polyP by the PPT/AdK system. Note that AMP released by certain ATP-dependent enzymes requires the activity of both PPT and AdK to regenerate ATP.

FIG. 2

Regeneration of ATP from polyP and AMP as demonstrated by sustained bioluminescence from firefly luciferase (assay 2). (A) Effect of assay constituents on ATP regeneration activity. Complete reaction mixtures contained the following, except where otherwise indicated: cell extract protein at 0.5 mg/ml, 3 mM MgCl₂, AdK at 1 U/ml, polyP at 0.2 g/liter, and 2 mM AMP. Symbols: □, 1.0 μM ATP; ○, 10 μM ATP; ▸, complete reaction mixture; ■, complete reaction mixture without AMP; ◃, complete reaction mixture without AdK; ×, complete reaction mixture without polyP. (B) Demonstration of the polyP requirement for ATP regeneration (assay constituents as defined for panel A). Symbols: ◊, 10 μM ATP; ○, complete reaction mixture; □, complete reaction mixture without polyP; ■, complete reaction mixture without polyP but with polyP at 0.2 g/liter added at 30 min.

FIG. 3

Substrate specificity of PPT. Reaction mixtures contained different nucleoside monophosphates (AMP, 2′-dAMP, GMP, CMP, UMP, and IMP) and were incubated with the 50% AS cut (0.23 mg of protein per ml; see Materials and Methods). The formation of the corresponding nucleoside di- and triphosphates was monitored by HPLC.

FIG. 4

Course of AMP (A) and 2′-dAMP (B) conversion to the corresponding nucleoside diphosphates (○) and triphosphates (▵) by the PPT/AdK system. Reaction mixtures were as described in the legend to Fig. 3 but contained 1.0 mM substrate and 0.46 mg of protein of a 50% AS cut per ml.

FIG. 5

HK-catalyzed formation of G6P in reaction mixtures with ATP supplied from 5 mM AMP and polyP_{n = 35} using AdK with different amounts of PPT protein. PPT concentrations, represented as total protein from a 40 to 60% AS cut, are 1.36 (■), 0.68 (⧫), 0.34 (●), and 0.136 (▴) mg/ml. Control reaction mixtures contained 5 mM ATP (dashed lines) with (○) and without (□) AdK.