Phosphoenolpyruvate synthase plays an essential role for glycolysis in the modified Embden-Meyerhof pathway in Thermococcus kodakarensis
Phosphoenolpyruvate synthase plays an essential role for glycolysis in the modified Embden-Meyerhof pathway in Thermococcus kodakarensis
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DOI：
10.1111/j.1365-2958.2006.05287.x
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发表时间：
2006-08-01
影响因子：
3.6
通讯作者：
Imanaka, Tadayuki
中科院分区：
文献类型：
--
作者：
Imanaka, Hiroyuki;Yamatsu, Atsushi;Imanaka, Tadayuki
我们对极端嗜热古菌Thermococcus kodakarensis中的丙酮酸激酶（Pyk（Tk））和磷酸烯醇式丙酮酸合酶（Pps（tk））进行了遗传分析。原则上，这两种酶都可以催化在嗜热球菌中发现的经修饰的Embden-Meyerhof（EM）途径的最后一步，即磷酸烯醇丙酮酸（PEP）转化为丙酮酸，前者利用ADP，而后者依赖于AMP和磷酸盐。Pyk（Tk）和Pps（Tk）的酶活性和转录水平在T.与在丙酮酸或氨基酸上生长的细胞相比，在糖酵解条件下的Kodakarens。使用具有trpE基因破坏的色氨酸营养缺陷型突变体KW 128作为宿主菌株，我们获得了在pyk（Tk）（Delta pyk菌株）或pps（Tk）（Delta pps菌株）基因中具有单基因破坏的突变菌株。在各种培养基中检查比生长速率和细胞产量，并与宿主KW 128菌株进行比较。结果表明，这两种酶都参与了丙酮酸的代谢，但不是必需的。在麦芽低聚糖的存在下，Delta pyk菌株的生长速率与宿主菌株相比降低了15%，这表明Pyk（Tk）确实参与糖酵解。然而，在Delta pps菌株中观察到甚至更显著的效果，因为该菌株根本不能在麦芽寡糖上生长。结果清楚地表明，与传统的依赖于丙酮酸激酶的EM途径相反，PEP合成酶是T.科达卡人。讨论了这两种酶在不同生长条件下的生理作用。
We have carried out a genetic analysis on pyruvate kinase (Pyk(Tk)) and phosphoenolpyruvate synthase (Pps(tk)) in the hyperthermophilic archaeon, Thermococcus kodakarensis. In principle, both enzymes can catalyse the final step of the modified Embden-Meyerhof (EM) pathway found in Thermococcales, the conversion of phosphoenolpyruvate (PEP) to pyruvate, with the former utilizing ADP, while the latter is dependent on AMP and phosphate. Enzyme activities and transcript levels of both Pyk(Tk) and Pps(Tk) increased in T. kodakarensis under glycolytic conditions when compared with cells grown on pyruvate or amino acids. Using KW128, a tryptophan auxotrophic mutant with a trpE gene disruption, as a host strain, we obtained mutant strains with single gene disruptions in either the pyk(Tk) (Delta pyk strain) or pps(Tk) (Delta pps strain) gene. Specific growth rates and cell yields were examined in various media and compared with the host KW128 strain. The results indicated that both enzymes participated in pyruvate metabolism, but were not essential. In the presence of maltooligosaccharides, the Delta pyk strain displayed a 15% decrease in growth rate compared with the host strain, indicating that Pyk(Tk) does participate in glycolysis. However an even more dramatic effect was observed in the Delta pps strain in that the strain could not grow at all on maltooligosaccharides. The results clearly indicate that, in contrast to the conventional EM pathway dependent on pyruvate kinase, PEP synthase is the essential enzyme for the glycolytic conversion of PEP to pyruvate in T. kodakarensis. The physiological roles of the two enzymes under various growth conditions are discussed.