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.
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 Mg2+-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.
Rébeillé
F
F
Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
Macherel
D
D
Mouillon
J M
JM
Garin
J
J
Douce
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004094:Dihydropteroate Synthase / Q000737:chemistry; D017854:Diphosphotransferases / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology*; D011622:Pterins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
C105176:6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-7,8-dihydropteroate synthase; D017931:DNA Primers; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D011622:Pterins; C000978:dihydropteroate; D004094:Dihydropteroate Synthase; D017854:Diphosphotransferases; C019684:2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase