CLONING, PURIFICATION, AND PROPERTIES OF A PHOSPHOTYROSINE PROTEIN PHOSPHATASE FROM STREPTOMYCES-COELICOLOR A3(2)

We describe the isolation and characterization of a gene (ptpA) from S treptomyces coelicolor A3(2) that codes for a protein with a deduced M (r) of 17,690 containing significant amino acid sequence identity with mammalian and prokaryotic small, acidic phosphotyrosine protein phosp hatases (PTPases). After expression of S. coelicolor ptpA in Escherich ia coli with a pT7-7-based vector system, PtpA was purified to homogen eity as a fusion protein containing five extra amino acids. The purifi ed fusion enzyme catalyzed the removal of phosphate from p-nitrophenyl phosphate (PNPP), phosphotyrosine (PY), and a commercial phosphopeptid e containing a single phosphotyrosine residue but did not cleave phosp hoserine or phosphothreonine. The pH optima for PNPP and PY hydrolysis by PtpA were 6.0 and 6.5, respectively. The K-m values for hydrolysis of PNPP and PY by PtpA were 0.75 mM (pH 6.0, 37 degrees C) and 2.7 mM (pH 6.5, 37 degrees C), respectively. Hydrolysis of PNPP by S. coelic olor PtpA was competitively inhibited by dephostatin with a K-i of 1.6 4 mu M; the known PTPase inhibitors phenylarsine oxide, sodium vanadat e, and iodoacetate also inhibited enzyme activity. Apparent homologs o f ptpA were detected in other streptomycetes by Southern hybridization ; the biological functions of PtpA and its putative homologs in strept omycetes are not yet known.