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Isolation and Sequence of the Phosphoenolpyruvate Carboxylase Gene of the Marine Cyanobacterium Synechococcus PCC 7002

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Author(s): A.A. Smith | M.W. Coomes | T.E. Smith

Journal: Journal of Biological Sciences
ISSN 1727-3048

Volume: 8;
Issue: 8;
Start page: 1261;
Date: 2008;
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Keywords: Synechococcus PCC 7002 | phosphoenolpyruvate carboxylase | pepc gene | cyanobacteria

ABSTRACT
The pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC), of the marine cyanobacterium Synechococcus PCC 7002, was isolated and sequenced. PEPC is an anaplerotic enzyme, but it may also contribute to overall CO2 fixation through β-carboxylation reactions. A consensus sequence generated by aligning the pepc genes of Anabaena variabilis, Anacystis nidulans and Synechocystis PCC 6803 was used to design two sets of primers that were used to amplify segments of Synechococcus PCC 7002 pepc. In order to isolate the gene, the sequence of the PCR product was used to search for the pepc nucleotide sequence from the publicly available incomplete genome of Synechococcus PCC 7002. Thus, the major challenge was to find the pepc gene among genomic fragments and to complete gaps as necessary. PCR primers were designed to amplify a DNA fragment using a high fidelity thermostable DNA polymerase. An Open Reading Frame (ORF) consisting of 2988 base pairs coding for 995 amino acids was found in the 3066 bp PCR product. The pepc gene had a GC content of 52% and the deduced protein had a calculated molecular mass of 114,049 Da. The amino acid sequence was closely related to that of PEPC from other cyanobacteria, exhibiting 59-61% identity. The sequence differed significantly from plant and E. coli PEPC with only 30% homology. However, most of the essential amino acids involved in PEPC activity were shared by both proteins. The recombinant Synechococcus PCC 7002 PEPC was expressed in E. coli.
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