Li Y, Strohl W R
Department of Microbiology, Ohio State University, Columbus 43210-1292, USA.
J Bacteriol. 1996 Jan;178(1):136-42. doi: 10.1128/jb.178.1.136-142.1996.
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 Km 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. coelicolor 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. coelicolor PtpA was competitively inhibited by dephostatin with a Ki of 1.64 microM; 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.
我们描述了从天蓝色链霉菌A3(2)中分离和鉴定一个基因（ptpA）的过程，该基因编码一种推导分子量为17,690的蛋白质，与哺乳动物和原核生物的小酸性磷酸酪氨酸蛋白磷酸酶（PTPases）具有显著的氨基酸序列同一性。在大肠杆菌中利用基于pT7-7的载体系统表达天蓝色链霉菌ptpA后，PtpA作为一种含有五个额外氨基酸的融合蛋白被纯化至同质。纯化的融合酶催化从对硝基苯磷酸酯（PNPP）、磷酸酪氨酸（PY）以及含有单个磷酸酪氨酸残基的商业磷酸肽上去除磷酸，但不切割磷酸丝氨酸或磷酸苏氨酸。PtpA水解PNPP和PY的最适pH分别为6.0和6.5。PtpA水解PNPP和PY的Km值分别为0.75 mM（pH 6.0，37℃）和2.7 mM（pH 6.5，37℃）。天蓝色链霉菌PtpA水解PNPP分别被去磷抑素竞争性抑制，Ki为1.64 μM；已知的PTPase抑制剂苯砷氧化物、钒酸钠和碘乙酸也抑制酶活性。通过Southern杂交在其他链霉菌中检测到了ptpA的明显同源物；PtpA及其在链霉菌中的假定同源物的生物学功能尚不清楚。
