Essential Role of the hprK Gene in Ralstonia eutropha H16
Essential Role of the hprK Gene in Ralstonia eutropha H16
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DOI：
10.1159/000233505
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发表时间：
2009-08
影响因子：
1.2
通讯作者：
Désirée Krauβe;K. Hunold;B. Kusian;O. Lenz;J. Stülke;B. Bowien;J. Deutscher
中科院分区：
文献类型：
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作者：
Désirée Krauβe;K. Hunold;B. Kusian;O. Lenz;J. Stülke;B. Bowien;J. Deutscher
富营养拉尔斯氏菌H16具有一个不完全的磷酸烯醇式丙酮酸(PEP)：由EI、HPR、EIIANtR(PtsN)和EIIAMAN(PtsM)组成的糖磷酸转移酶系统(PTS)。我们可以证明，在体外，不完全的PTS磷酸化级联是部分功能的。HPR被PEP和EI磷酸化，并将磷酰基转移到EIIANtR，但转移到EIIAMAN的速度非常慢。该系统的组成部分先前已被证明调节聚羟基丁酸酯的新陈代谢。在ptsN下游，该生物含有hprK基因，该基因编码hpr激酶/磷酸化酶的同源物。我们发现该酶以三磷酸腺苷为磷酸化供体，使HPR磷酸化。有趣的是，hprK似乎在富营养化杆菌中是必不可少的，因为这个基因在野生型菌株中不能被删除，但在缺乏ptsH或ptsi的突变体中可以被删除。这表明hpr和/或P∼His-hpr浓度的增加可能是导致生长缺陷的原因。为了验证这一假设，在ptsH hprK双突变体中引入了不同的ptsH等位基因。该突变体只能与ptsH(His15Ala)等位基因互补，而不能与野生型或ptsH(Ser46Ala)等位基因互补。我们得出结论，hprK突变体中形成的高数量的His-15-磷酸化HPR是其生长缺陷的原因。
Ralstonia eutropha H16 possesses an incomplete phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) composed of EI, HPr, EIIANtr (PtsN) and EIIAMan (PtsM). We could show that in vitro the incomplete PTS phosphorylation cascade is partially functional. HPr becomes phosphorylated by PEP and EI, and transfers the phosphoryl group to EIIANtr, but only extremely slowly to EIIAMan. Components of this system have previously been shown to regulate the metabolism of polyhydroxybutyrate. Downstream from ptsN this organism contains an hprK gene, which codes for a homologue of HPr kinase/phosphorylase. We show that this enzyme phosphorylates HPr using ATP as phosphoryl donor. Interestingly, hprK appeared to be essential in R. eutropha because this gene could not be deleted in the wild-type strain, but could be deleted in mutants lacking ptsH or ptsI. This suggests that an increase in the HPr and/or P∼His-HPr concentrations might be responsible for the growth defect. To test this hypothesis, various ptsH alleles were introduced into the ptsH hprK double mutant. Complementation of this mutant was possible only with the ptsH(His15Ala) allele, but not with the wild-type or ptsH(Ser46Ala) alleles. We conclude that elevated amounts of His-15-phosphorylated HPr, formed in the hprK mutant, are responsible for its growth defect.