Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2)

Abstract

We describe the isolation and characterization of a gene (ptpA) from Streptomyces 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 phosphatases (PTPases). After expression of S. coelicolor ptpA in Escherichia coli with a pT7-7-based vector system, PtpA was purified to homogeneity as a fusion protein containing five extra amino acids. The purified fusion enzyme catalyzed the removal of phosphate from p-nitrophenylphosphate (PNPP), phosphotyrosine (PY), and a commercial phosphopeptide containing a single phosphotyrosine residue but did not cleave phosphoserine 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؇C) and 2.7 mM (pH 6.5, 37؇C), respectively. Hydrolysis of PNPP by S. coelicolor PtpA was competitively inhibited by dephostatin with a K i of 1.64 M; the known PTPase inhibitors phenylarsine oxide, sodium vanadate, and iodoacetate also inhibited enzyme activity. Apparent homologs of ptpA were detected in other streptomycetes by Southern hybridization; the biological functions of PtpA and its putative homologs in streptomycetes are not yet known.Streptomycetes are gram-positive, aerobic, mycelium-forming bacteria that undergo a complex morphological differentiation that includes the sequential production of substrate mycelium, aerial mycelium, and spores (9). Concomitant with morphological differentiation, these organisms undergo a biochemical differentiation during which they produce economically important secondary metabolites. Several studies have been carried out indicating that protein phosphorylation takes place in Streptomyces coelicolor and other streptomycetes (14,33,42,47), and a protein kinase that phosphorylates serine and threonine residues in vitro was discovered recently (33). Using monoclonal anti-phosphotyrosine (PY) antibodies 4G10 and IG2, Waters et al. (47) recently showed evidence for protein tyrosine phosphorylation in Streptomyces hygroscopicus, Streptomyces griseus, Streptomyces lividans, and S. coelicolor. Furthermore, some unidentified protein tyrosine kinase activities were also observed in extracts of these strains (47), suggesting the presence of proteins in streptomycetes that contain PY residues. We report here the isolation of a gene (ptpA) from S. coelicolor A3(2) encoding a small, acidic PY protein phosphatase (SA-PTPase) and characterization of its product, PtpA, which has several characteristics similar to those of homologs characterized from mammalian systems. Although PtpA was shown to possess PTPase activity in vitro, its biological function in S. coelicolor is still unknown. MATERIALS AND METHODSBacterial strains and plasmids. Streptomyces azureus, Streptomyces peucetius, and Streptomyces insignis were obtained from the American Type Culture Collection as ATCC strains 14921, 29050, and 31913, respectiv...

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A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

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