Staphylococcus aureus contains two low-molecular-mass phosphotyrosine protein phosphatases

Abstract

The analysis of the different amino acid sequences deduced from the complete genome sequence of the gram-positive bacterium Staphylococcus aureus suggested the presence of two eukaryotic-protein-like low-molecular-mass phosphotyrosine protein phosphatases, which are usually found in gram-negative bacteria. To check this prediction, the corresponding genes were cloned and overexpressed in an Escherichia coli system. Two distinct proteins with an apparent molecular mass of 23 kDa each, PtpA and PtpB, were produced and then purified by affinity chromatography and assayed for enzymatic properties. As expected, they both exhibited phosphatase activity in vitro, with a maximum value at a pH of around 6.2 and at a temperature of 40 degrees C. In addition, their kinetic constants, their specificity for phosphotyrosine residues, and their sensitivity to two phosphatase inhibitors, N-ethylmaleimide and orthovanadate, matched those of acid low-molecular-mass phosphotyrosine protein phosphatases.

FIG. 1.

Comparison of PtpA and PtpB with different prokaryotic and eukaryotic low-molecular-mass phosphotyrosine protein phosphatases. The alignment was made by using the CLUSTAL W program (28). Asterisks indicate the amino acid residues identical in all proteins, and underlining indicates similarity. The typical signatures of phosphotyrosine protein phosphatases are framed (C-X₄-C-R and D-P-Y). The amino acid numbering is based on the amino acid sequence of protein PtpA.

FIG. 2.

Overproduction and purification of PtpA and PtpB. The soluble fraction from BL21(DE3) (pET15b-ptpA) cells grown for 2 h at 37°C in the absence (lane 1) or in the presence (lane 2) of 1 mM IPTG was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue. Protein PtpA was purified by affinity chromatography on Ni-NTA agarose (lane 3). The soluble fraction from BL21(DE3) (pREP4-groESL) (pET15b-ptpB) cells grown for 3 h at 20°C in the absence (lane 4) or in the presence (lane 5) of 1 mM IPTG was analyzed under the same conditions. Protein PtpB was purified by affinity chromatography on Ni-NTA agarose (lane 6).

FIG. 3.

Phosphatase activity of PtpA and PtpB as a function of pH and temperature. The phosphatase activity was measured by using 40 mM PNPP as a substrate and 0.4 μg of PtpA (○) or PtpB (□) per assay. (A) pH varying from 5.0 to 6.75; (B) temperature varying from 20 to 50°C.

FIG. 4.

Effect of phosphatase inhibitors on the activity of PtpA and PtpB. The phosphatase activity was measured by using 40 mM PNPP as a substrate and 0.4 μg of PtpA (○) or PtpB (□) per assay. Two different inhibitors of phosphotyrosine phosphatases were used at the indicated concentrations. The results are expressed in each case as a percentage of the phosphatase activity measured in the absence of inhibitor (taken as 100%).