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.