A phosphotransacetylase from uropathogenic Staphylococcus saprophyticus: Characterization, kinetic analysis and its allosteric inhibition by ?-ketoglutarate

Abstract

Objectives: The genome of Staphylococcus saprophyticus, an uropathogen, contains SSP2124 gene encoding a putative phosphotransacetylase (Pta). Pta plays significant role in energy metabolism as well as colonization of uropathogens. In this study, cloning, expression, characterization and kinetic analysis of S. saprophyticus Pta have been undertaken. Methods: The SSP2124 gene was amplified by PCR from the genome of S. saprophyticus and subcloned into pET28a(+) vector and expressed in E. coli BL21. The recombinant Pta was affinity purified by Ni-NTA column. Its characterization as well as its steady-state kinetic analysis has been studied by standard procedures. Results: The recombinant Pta forms dimers as determined by gel filtration. The activity of the enzyme was maximal at a temperature range of 30-35 degrees C and at the pH range of 7.5-8.5. It was greatly stimulated by K+ and NH4+ ions, but inhibited by Na+ ion. Kinetic analysis and product inhibition studies suggest that there is a kinetic mechanism that proceeds through random addition of both substrates to the enzyme before any product is released. The enzyme was inhibited in both acetyl-CoA-forming (forward) and acetyl phosphate-forming (reverse) directions by adenosine triphosphate (ATP) and alpha-ketoglutarate but not affected by pyruvate and reduced nicotinamide adenine dinucleotide. ATP was competitive with respect to CoA and noncompetitive with respect to acetyl phosphate. The inhibition by alpha-ketoglutarate, on the other hand, was allosteric with half-saturations at 6.5 +/- 0.7 mM and Hill coefficients of 2.6 +/- 0.8 and 3.3 +/- 0.2, in forward and reverse directions, respectively. Conclusions: S. saprophyticus Pta belongs to the Pta I family and show similar biochemical features as well as kinetic mechanism with other members. And the most importantly, this study presents the first example of allosteric regulation of a member of class I Ptas.