Journal Article

Molecular Characterization of a Phosphoenolpyruvate Carboxylase from a Thermophilic Cyanobacterium, <i>Synechococcus vulcanus</i> with Unusual Allosteric Properties

Li-mei Chen, Takuma Omiya, Shingo Hata and Katsura Izui

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 43, issue 2, pages 159-169
Published in print February 2002 | ISSN: 0032-0781
Published online February 2002 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcf019
Molecular Characterization of a Phosphoenolpyruvate Carboxylase from a Thermophilic Cyanobacterium, Synechococcus vulcanus with Unusual Allosteric Properties

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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°C for 60 min or exposure to 0.5 M guanidine-HCl at 30°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°C at pH 7.5 and an optimum pH of 9.0 at 30°C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P2), 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. I0.5 for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P2, Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.

Keywords: Key words: Heat stability — Phosphoenolpyruvate carboxylase (EC 4.1.1.31) — Recombinant protein — Regulatory properties —Synechococcus vulcanus — Thermophilic cyanobacterium.; Abbreviations: Fruc-1,6-P2, fructose-1,6-bisphosphate; Gdn-HCl, guanidine hydrochloride; Gluc-6-P, glucose 6-phosphate; IPTG, isopropyl-β-d-thiogalactopyranoside; LB, Luria-Bertani; MDH, malate dehydrogenase; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; TCA, tricarboxylic acid.

Journal Article.  6767 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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