Molecular characterization of a phosphoenolpyruvate carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties.
A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe. The S. vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa. SvPEPC was expressed in E. coli BL21 Codonplus (DE3), using pET32a as a vector. The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE. The enzyme forms homotetramer as judged by gel filtration. SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)). SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln. Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.
Chen
Li-mei
LM
Laboratory of Plant Physiology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan.
Omiya
Takuma
T
Hata
Shingo
S
Izui
Katsura
K
D000494:Allosteric Regulation / Q000187:drug effects; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000037:antagonists & inhibitors / Q000494:pharmacology; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005635:Fructosediphosphates / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D015698:Genomic Library; D005988:Glyceric Acids / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008293:Malates / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010730:Phosphoenolpyruvate Carboxylase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D005635:Fructosediphosphates; D005988:Glyceric Acids; D008293:Malates; D001224:Aspartic Acid; D010728:Phosphoenolpyruvate; C030298:malic acid; C005156:3-phosphoglycerate; D010730:Phosphoenolpyruvate Carboxylase; C029063:fructose-1,6-diphosphate