Cloning and Characterization of Mitochondrial 5-Formyltetrahydrofolate Cycloligase from Higher Plants
Copyright policy )Cloning and Characterization of Mitochondrial 5-Formyltetrahydrofolate Cycloligase from Higher Plants
5-Formyltetrahydrofolate cycloligase (5-FCL) catalyzes the conversion of 5-formyltetrahydrofolate (5-CHO-H(4)PteGlu(n)) to 5,10-methenyltetrahydrofolate and is considered to be the main means whereby 5-CHO-H(4)PteGlu(n) is metabolized in mammals, yeast, and bacteria. 5-CHO-H(4)PteGlu(n) is known to occur in plants and to be highly abundant in leaf mitochondria. Genomics-based approaches identified Arabidopsis and tomato cDNAs encoding proteins homologous to 5-FCLs of other organisms but containing N-terminal extensions with the features of mitochondrial targeting peptides. These homologs were shown to have 5-FCL activity by characterizing recombinant enzymes produced in Escherichia coli and by functional complementation of a yeast fau1 mutation with the Arabidopsis 5-FCL cDNA. The recombinant Arabidopsis enzyme is active as a monomer, prefers the penta- to the monoglutamyl form of 5-CHO-H(4)PteGlu(n), and has kinetic properties broadly similar to those of 5-FCLs from other organisms. Enzyme assays and immunoblot analyses indicated that 5-FCL is located predominantly if not exclusively in plant mitochondria and that the mature, active enzyme lacks the putative targeting sequence. Serine hydroxymethyltransferase (SHMT) from plant mitochondria was shown to be inhibited by 5-CHO-H(4)PteGlu(n) as are SHMTs from other organisms. Since mitochondrial SHMT is crucial to photorespiration, 5-FCL may help prevent 5-CHO-H(4)PteGlu(n) from reaching levels that would inhibit this process. Consistent with this possibility, 5-FCL activity was far higher in leaf mitochondria than root mitochondria.
- Washington State University United States
- University of Florida United States
570, DNA, Complementary, Molecular Sequence Data, Complementary - genetics, Arabidopsis, Arabidopsis - enzymology, Sequence Homology, Carbon-Nitrogen Ligases - genetics, Substrate Specificity, Solanum lycopersicum, Recombinant Proteins - metabolism, Escherichia coli, Animals, Humans, Carbon-Nitrogen Ligases, Amino Acid Sequence, Cloning, Molecular, Phylogeny, DNA Primers, 580, Base Sequence, Sequence Homology, Amino Acid, Carbon-Nitrogen Ligases - metabolism, Molecular, DNA, Recombinant Proteins, Mitochondria, Molecular Weight, Amino Acid, Escherichia coli - enzymology, Mitochondria - enzymology, Lycopersicon esculentum - genetics, Sequence Alignment, Escherichia coli - genetics, Cloning
570, DNA, Complementary, Molecular Sequence Data, Complementary - genetics, Arabidopsis, Arabidopsis - enzymology, Sequence Homology, Carbon-Nitrogen Ligases - genetics, Substrate Specificity, Solanum lycopersicum, Recombinant Proteins - metabolism, Escherichia coli, Animals, Humans, Carbon-Nitrogen Ligases, Amino Acid Sequence, Cloning, Molecular, Phylogeny, DNA Primers, 580, Base Sequence, Sequence Homology, Amino Acid, Carbon-Nitrogen Ligases - metabolism, Molecular, DNA, Recombinant Proteins, Mitochondria, Molecular Weight, Amino Acid, Escherichia coli - enzymology, Mitochondria - enzymology, Lycopersicon esculentum - genetics, Sequence Alignment, Escherichia coli - genetics, Cloning
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