HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Takai, K. Articles by Uchida, A. Search for Related Content PubMed PubMed Citation Articles by Takai, K. Articles by Uchida, A. Agricola Articles by Takai, K. Articles by Uchida, A.

ppc, the gene for phosphoenolpyruvate carboxylase from an extremely thermophilic bacterium, Rhodothermus obamensis: cloning, sequencing and overexpression in Escherichia coli

Ken Takai^1,*,

Laboratory of Marine Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

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.

^*Author for correspondence: Ken Takai. Tel: + 81 468 67 3894. Fax: +81 468 66 6364. e-mail: kent@jamstec.go.jp

Present address: Deep-sea Microorganisms Research Group (DEEP-STAR), Japan Marine Science and Technology Center (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237, Japan.