PPC, THE GENE FOR PHOSPHOENOLPYRUVATE CARBOXYLASE FROM AN EXTREMELY THERMOPHILIC BACTERIUM, RHODOTHERMUS-OBAMENSIS - CLONING, SEQUENCING AND OVEREXPRESSION IN ESCHERICHIA-COLI

The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, Rhodothermus obamensis, was direc tly 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 c alculated molecular mass was 107848 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 o f PEPC from another thermophile, a Thermus sp., and from a mesophile, Corynebacterium glutamicum, exhibiting 45.3 % or 37.7 % identity and 6 1.5 % or 56.5 % similarity, respectively. By Southern analysis, the pp c 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 BET expression vector system and a thermostable recombinant PEPC wa s obtained. Comparison of the deduced amino acid sequences of the ther mophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two therm ophilic enzymes.