Shi L, Carmichael W W, Kennelly P J
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
J Biol Chem. 1999 Apr 9;274(15):10039-46. doi: 10.1074/jbc.274.15.10039.
The structural gene for a putative PPP family protein-serine/threonine phosphatase from the microcystin-producing cyanobacterium Microcystis aeruginosa PCC 7820, pp1-cyano1, was cloned. The sequence of the predicted gene product, PP1-cyano1, was 98% identical to that of the predicted product of an open reading frame, pp1-cyano2, from a cyanobacterium that does not produce microcystins, M. aeruginosa UTEX 2063. By contrast, PP1-cyano1 displayed less than 20% identity with other PPP family protein phosphatases from eukaryotic, archaeal, or other bacterial organisms. PP1-cyano1 and PP1-cyano2 were expressed in Escherichia coli and purified to homogeneity. Both enzymes exhibited divalent metal dependent phosphohydrolase activity in vitro toward phosphoserine- and phosphotyrosine-containing proteins and 3-phosphohistidine- and phospholysine-containing amino acid homopolymers. This multifunctional potential also was apparent in samples of PP1-cyano1 and PP1-cyano2 isolated from M. aeruginosa. Catalytic activity was insensitive to okadaic acid or the cyanobacterially produced cyclic heptapeptide, microcystin-LR, both potent inhibitors of mammalian PP1 and PP2A. PP1-cyano1 and PP1-cyano2 displayed diadenosine tetraphosphatase activity in vitro. Diadenosine tetraphosphatases share conserved sequence features with PPP family protein phosphatases. The diadenosine tetraphosphatase activity of PP1-cyano1 and PP1-cyano2 confirms that these enzymes share a common catalytic mechanism.
克隆了来自产微囊藻毒素的蓝藻铜绿微囊藻PCC 7820的一种假定的PPP家族蛋白丝氨酸/苏氨酸磷酸酶的结构基因pp1-cyano1。预测的基因产物PP1-cyano1的序列与来自不产微囊藻毒素的蓝藻铜绿微囊藻UTEX 2063的一个开放阅读框pp1-cyano2的预测产物的序列有98%的同一性。相比之下，PP1-cyano1与来自真核生物、古细菌或其他细菌的其他PPP家族蛋白磷酸酶的同一性不到20%。PP1-cyano1和PP1-cyano2在大肠杆菌中表达并纯化至同质。两种酶在体外均表现出对含磷酸丝氨酸和磷酸酪氨酸的蛋白质以及含3-磷酸组氨酸和磷酸赖氨酸的氨基酸均聚物的二价金属依赖性磷酸水解酶活性。这种多功能潜力在从铜绿微囊藻分离的PP1-cyano1和PP1-cyano2样品中也很明显。催化活性对冈田酸或蓝藻产生的环状七肽微囊藻毒素-LR不敏感，这两种物质都是哺乳动物PP1和PP2A的有效抑制剂。PP1-cyano1和PP1-cyano2在体外表现出二腺苷四磷酸酶活性。二腺苷四磷酸酶与PPP家族蛋白磷酸酶具有保守的序列特征。PP1-cyano1和PP1-cyano2的二腺苷四磷酸酶活性证实这些酶具有共同的催化机制。
