microbiology, Vol 144, 1423-1434, Copyright © 1998 by Society for General Microbiology
ppc, the gene for phosphoenolpyruvate carboxylase from an extremely thermophilic bacterium, Rhodothermus obamensis: cloning, sequencing and overexpression in Escherichia coli
K Takai, Y Sako and A Uchida
Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Japan. kent@jamstec.go.jp
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.
