Polyphosphate kinase of Acinetobacter sp strain ADP1: Purification and characterization of the enzyme and its role during changes in extracellular phosphate levels

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabol ism are incompletely understood. The polyphosphate kinase (PPK) of Acinetob acter sp. strain ADP1, an organism that accumulates large amounts of polyp, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monom er, PPK is sensitive to magnesium concentrations, and optimum activity occu rs in the presence. of 3 mM MgCl2. The optimum pH was between pH 7 and 8, a nd significant reductions in activity occurred at lower pH values. The grea test activity occurred at 40 degrees C. The half-saturation ATP concentrati on for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyp mono mers per mu g of protein per min, PPK was the primary, although not the sol e, enzyme responsible for the production of polyP in Acinetobacter sp. stra in ADP1. Under low-phosphate (P-i) conditions, despite strong induction of the ppk gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in Acinetobacter sp. strain ADP1. Once exce ss phosphate was added to the P-i-starved culture, both the polyp synthesis activity and the levels of polyP rose sharply. Increases in polyP-degradin g activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P-i cond itions. This activity declined when phosphate was added.