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Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase/7,8‐dihydropteroate synthase localized in mitochondria
Author(s) -
Rébeillé Fabrice,
Macherel David,
Mouillon JeanMarie,
Garin Jérome,
Douce Roland
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.5.947
Subject(s) - biology , dihydropteroate synthase , transit peptide , biochemistry , peptide sequence , atp synthase , phosphofructokinase 2 , amino acid , biosynthesis , enzyme , complementary dna , mitochondrial matrix , microbiology and biotechnology , chloroplast , gene , cytosol , plasmodium falciparum , pyrimethamine , malaria , immunology , plastid
In pea leaves, the synthesis of 7,8‐dihydropteroate, a primary step in folate synthesis, was only detected in mitochondria. This reaction is catalyzed by a bifunctional 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase/7,8‐dihydropteroate synthase enzyme, which represented 0.04–0.06% of the matrix proteins. The enzyme had a native mol. wt of 280–300 kDa and was made up of identical subunits of 53 kDa. The reaction catalyzed by the 7,8‐dihydropteroate synthase domain of the protein was Mg 2+ ‐dependent and behaved like a random bireactant system. The related cDNA contained an open reading frame of 1545 bp and the deduced amino acid sequence corresponded to a polypeptide of 515 residues with a calculated M r of 56 454 Da. Comparison of the deduced amino acid sequence with the N‐terminal sequence of the purified protein indicated that the plant enzyme is synthesized with a putative mitochondrial transit peptide of 28 amino acids. The calculated M r of the mature protein was 53 450 Da. Southern blot experiments suggested that a single‐copy gene codes for the enzyme. This result, together with the facts that the protein is synthesized with a mitochondrial transit peptide and that the activity was only detected in mitochondria, strongly supports the view that mitochondria is the major (unique?) site of 7,8‐dihydropteroate synthesis in higher plant cells.