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

EMBO J. 1997 Mar 3;16(5):947-57. doi: 10.1093/emboj/16.5.947.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Blotting, Northern
  • Chromatography, Gel
  • Cloning, Molecular
  • DNA Primers
  • Dihydropteroate Synthase / chemistry
  • Diphosphotransferases / chemistry
  • Electrophoresis, Agar Gel
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Inhibitors / pharmacology
  • Kinetics
  • Mitochondria / enzymology*
  • Molecular Sequence Data
  • Molecular Weight
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism
  • Peas / enzymology*
  • Pterins / metabolism*
  • Sequence Homology, Amino Acid

Substances

  • 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-7,8-dihydropteroate synthase
  • DNA Primers
  • Enzyme Inhibitors
  • Multienzyme Complexes
  • Pterins
  • dihydropteroate
  • Dihydropteroate Synthase
  • Diphosphotransferases
  • 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase

Associated data

  • GENBANK/Y08611