Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli.
The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.
Nast
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G
Institut für Genbiologische Forschung Berlin GmbH, Germany.
Müller-Röber
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B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D005649:Fumarate Hydratase / Q000096:biosynthesis* / Q000737:chemistry*; D006801:Humans; D008099:Liver / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018515:Plant Leaves; D018517:Plant Roots; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D011994:Recombinant Proteins; D005649:Fumarate Hydratase