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Phosphoenolpyruvate synthase plays an essential role for glycolysis in the modified Embden‐Meyerhof pathway in Thermococcus kodakarensis
Author(s) -
Imanaka Hiroyuki,
Yamatsu Atsushi,
Fukui Toshiaki,
Atomi Haruyuki,
Imanaka Tadayuki
Publication year - 2006
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2006.05287.x
Subject(s) - phosphoenolpyruvate carboxykinase , biology , thermococcus , pyruvate kinase , glycolysis , biochemistry , enzyme , mutant , gene , archaea
Summary 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 ( Δpyk strain) or pps Tk ( Δpps 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 Δpyk 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 Δpps 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.