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 pyrophos phokinase/7,8-dihydropteroate synthase enzyme, which represented 0.04- 0.06% of the matrix proteins. The enzyme had a native mel. wt of 280-3 00 kDa and was made up of identical subunits of 53 kDa. The reaction c atalyzed by the 7,8-dihydropteroate synthase domain of the protein was Mg2+-dependent and behaved like a random bireactant system. The relat ed cDNA contained an open reading frame of 1545 bp and the deduced ami no acid sequence corresponded to a polypeptide of 515 residues with a calculated M(r) of 56 454 Da. Comparison of the deduced amino acid seq uence with the N-terminal sequence of the purified protein indicated t hat the plant enzyme is synthesized with a putative mitochondrial tran sit peptide of 28 amino acids, The calculated M(r) of the mature prote in was 53 450 Da. Southern blot experiments suggested that a single-co py gene codes for the enzyme. This result, together with the facts tha t 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-dihydro pteroate synthesis in higher plant cells.