title: Allosteric Inhibition of Phosphoenolpyruvate Carboxylases is Determined by a Single Amino Acid Residue in Cyanobacteria.
abstract: Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme for CO2fixation and primary metabolism in photosynthetic organisms including cyanobacteria. The kinetics and allosteric regulation of PEPCs have been studied in many organisms, but the biochemical properties of PEPC in the unicellular, non-nitrogen-fixing cyanobacterium Synechocystis sp. PCC 6803 have not been clarified. In this study, biochemical analysis revealed that the optimum pH and temperature of Synechocystis 6803 PEPC proteins were 7.3 and 30 °C, respectively. Synechocystis 6803 PEPC was found to be tolerant to allosteric inhibition by several metabolic effectors such as malate, aspartate, and fumarate compared with other cyanobacterial PEPCs. Comparative sequence and biochemical analysis showed that substitution of the glutamate residue at position 954 with lysine altered the enzyme so that it was inhibited by malate, aspartate, and fumarate. PEPC of the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 was purified, and its activity was inhibited in the presence of malate. Substitution of the lysine at position 946 (equivalent to position 954 in Synechocystis 6803) with glutamate made Anabaena 7120 PEPC tolerant to malate. These results demonstrate that the allosteric regulation of PEPC in cyanobacteria is determined by a single amino acid residue, a characteristic that is conserved in different orders.
authors.affiliation: School of Agriculture, Meiji University, 1-1-1, Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeya
authors.affiliation: RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Masami Yokota
authors.initials: MY
authors.lastname: Hirai
authors.affiliation: School of Agriculture, Meiji University, 1-1-1, Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.|RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Osanai
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors; D007700:Kinetics; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D046939:Synechocystis / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D010730:Phosphoenolpyruvate Carboxylase