Jones G H, Bibb M J
Department of Biology, Emory University, Atlanta, Georgia 30322, USA.
J Bacteriol. 1996 Jul;178(14):4281-8. doi: 10.1128/jb.178.14.4281-4288.1996.
The gene for the enzyme guanosine pentaphosphate synthetase I (GPSI) from Streptomyces antibioticus has been cloned and sequenced. The cloned gene functioned as a template in the streptomycete coupled transcription-translation system and directed the synthesis of a protein with the properties expected for GPSI. Sequencing of the cloned gene identified an open reading frame of 740 amino acids whose amino terminal sequence corresponded to the N terminus of purified GPSI. The GPSI protein sequence was found to possess significant homology to polynucleotide phosphorylase from Escherichia coli. Indeed, like E. coli polynucleotide phosphorylase, purified GPSI was shown to catalyze the polymerization of ADP and the phosphorolysis of poly(A). However, the E. coli enzyme was unable to catalyze the synthesis of guanosine pentaphosphate under conditions in which GPSI was highly active in that reaction. Overexpression of the cloned gpsI gene in E. coli led to an increase in both polynucleotide phosphorylase and guanosine pentaphosphate synthetase activities in the cloning host. The polynucleotide phosphorylase activities of GPSI and of the E. coli enzyme were strongly inhibited by dCDP, but the pppGpp synthetase activity of GPSI was not inhibited and indeed was slightly stimulated by dCDP. These results strongly support the identity of GPSI as a bifunctional enzyme capable of both pppGpp synthesis and polynucleotide phosphorylase activities.
来自抗生链霉菌的鸟苷五磷酸合成酶I（GPSI）基因已被克隆和测序。克隆的基因在链霉菌偶联转录-翻译系统中作为模板发挥作用，并指导合成具有GPSI预期特性的蛋白质。对克隆基因的测序确定了一个740个氨基酸的开放阅读框，其氨基末端序列与纯化的GPSI的N末端相对应。发现GPSI蛋白序列与大肠杆菌的多核苷酸磷酸化酶具有显著的同源性。实际上，与大肠杆菌多核苷酸磷酸化酶一样，纯化的GPSI被证明能催化ADP的聚合和聚（A）的磷酸解。然而，在GPSI在该反应中高度活跃的条件下，大肠杆菌酶无法催化鸟苷五磷酸的合成。在大肠杆菌中克隆的gpsI基因的过表达导致克隆宿主中多核苷酸磷酸化酶和鸟苷五磷酸合成酶活性均增加。GPSI和大肠杆菌酶的多核苷酸磷酸化酶活性受到dCDP的强烈抑制，但GPSI的pppGpp合成酶活性不受抑制，实际上还受到dCDP的轻微刺激。这些结果有力地支持了GPSI作为一种具有pppGpp合成和多核苷酸磷酸化酶活性的双功能酶的身份。
