title: ppc, the gene for phosphoenolpyruvate carboxylase from an extremely thermophilic bacterium, Rhodothermus obamensis: cloning, sequencing and overexpression in Escherichia coli.
abstract: The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, Rhodothermus obamensis, was directly sequenced by the thermal asymmetric interlaced (TAIL) PCR method. An ORF for a 937 amino acid polypeptide was found in the gene. The ppc gene had a high G+C content (66.2 mol%) and the third position of the codon exhibited strong preference for G or C usage (85.0 mol%). The calculated molecular mass was 107,848 Da, which was consistent with the molecular mass of the enzyme as determined by SDS-PAGE (100 kDa). The amino acid sequence of R. obamensis PEPC was closely related to that of PEPC from another thermophile, a Thermus sp., and from a mesophile, Corynebacterium glutamicum, exhibiting 45.3% or 37.7% identity and 61.5% or 56.5% similarity, respectively. By Southern analysis, the ppc gene was found to be present in a single copy in the genomic DNA of this organism. The cloned gene was expressed in Escherichia coli using a pET expression vector system and a thermostable recombinant PEPC was obtained. Comparison of the deduced amino acid sequences of the thermophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two thermophilic enzymes.
authors.affiliation: Laboratory of Marine Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Takai
authors.affiliation: Laboratory of Marine Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Sako
authors.affiliation: Laboratory of Marine Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Aritsune
authors.initials: A
authors.lastname: Uchida
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000032:analysis; D041963:Bacteroidetes / Q000201:enzymology* / Q000235:genetics; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA
chemical_list: D000596:Amino Acids; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010730:Phosphoenolpyruvate Carboxylase