Jean-Marie Mouillon
Study of folate metabolism in plants: characterization of the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase
Published on 27 January 2000
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Thesis presented January 27, 2000
Abstract:
One-carbon metabolism in cells is mediated by a variety of tetrahydrofolate polyglutamate derivatives. A number of pathways such as those involved in the metabolism of methionine, serine, purine or thymidylate synthesis are dependent on an endogenous supply of these coenzymes. Plants and micro-organisms, in contrast to animals, are able to synthesize tetrahydrofolate
de novo. This pathway requires the sequential operation of five enzymes which are mainly localized in plant mitochondria. In pea leaf mitochondria, the synthesis of 7,8-dihydropteroate, a primary step in folate synthesis, is catalyzed by the bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase enzyme (HPPK-DHPS). This bifunctional protein have been purified from pea leaf mitochondria and represents only 0.04-0.06 % of the matrix protein.
We have cloned the cDNA encoding for the HPPK-DHPS, and comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein indicates that the plant enzyme is synthesized with a putative mitochondrial transit peptide. In addition, Southern blot experiments suggested that a single-copy gene codes for the enzyme.
The reaction catalyzed by the DHPS domain behaves like a random bireactant system, and DHPS appears as an important regulatory point of the folate biosynthetic pathway. Indeed, dihydropteroate, dihydrofolate and tetrahydrofolate are strong inhibitors of DHPS activity. This activity is also the target of sulfonamide drugs and is therefore a potential target for herbicides. Biochemical properties of the HPPK domain indicate a sequential ordered system and a regulation by para-aminobenzoic acid, the substrate of DHPS activity. These last result indicates that the two reactions catalyzed by this bifunctional protein are tightly coupled.
In plant cells, two reactions are potentially important to provide the C1 units required in C1 metabolism. They are the serine hydroxymethyltransferase (SHMT) and the glycine decarboxylase (GDC). Although the GDC is mostly present in leaf tissues, we observed that it also plays a very important role in non-photosynthetic tissues during serine and glycine catabolism. Serine catabolism in the cytosol is the main source of the C1 units but the resulting glycine is oxidized in mitochondria, thus forming a "serine-glycine" cycle.
Keywords:
Plant mitochondria, C1 metabolism, Folate biosynthetic pathway, Dihydropteroate synthase, Serine and glycine catabolism
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