Essay 11.8

Coenzyme Synthesis in Plant Mitochondria

Stéphane Ravanel, Claude Alban, and Fabrice Rébeillé
Laboratoire de Physiologie Cellulaire Végétale, UMR5168 CNRS-CEA-INRA-Université Joseph Fourier Grenoble I, institut de Recherches en Sciences et Technologies pour le Vivant, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France

September, 2010

Introduction

The synthesis of many biological compounds and the regulation of several metabolic processes require the addition or removal of one-carbon units. Tetrahydrofolate derivatives (vitamin B9) mediate most of these transfers of C1-units, directly or indirectly via S-adenosylmethionine. Some enzymes involved in CO₂ manipulation during carboxylation, decarboxylation and transcarboxylation reactions utilize another essential cofactor, biotin (vitamin B8). Unlike plants and some fungi and bacteria, animals cannot synthesize tetrahydrofolate or biotin de novo and depend entirely on their dietary supply. The aim of the present Essay is to summarize recent progress in elucidating the tetrahydrofolate and biotin biosynthetic pathways in plants, with emphasis on the essential role of mitochondria in these complex networks.

Mitochondria Play an Essential Role in Tetrahydrofolate Synthesis

Structure and functions of folates
Tetrahydrofolate (THF) and its derivatives, collectively termed folates, are tripartite molecules with a pterin ring, a para-aminobenzoate (PABA) moiety, and a glutamate chain with 1 to 8 residues (Figure 1; Scott et al. 2000; Hanson and Roje 2001). The physiological forms of folates are mainly polyglutamylated, and folate-dependent enzymes prefer these coenzymes. The THF-carried C1-units exist in various oxidation states - from the most oxidized formyl to the most reduced methyl levels - and are attached to THF at positions N5 of the pterin ring, N10 of the PABA moiety, or as a bridge between the two (Figure 1). One remarkable feature of folate metabolism is its ability to interconvert the C1-units at different oxidation levels. Folates are involved in several key cellular processes including the synthesis of nucleic acids (purines, thymidylate), some amino acids (methionine, glycine, serine), formylmethionine-tRNA, pantothenate (vitamin B5), and, specific to plants, in photorespiration.

Figure 1 Chemical structures of folate-coenzymes. Natural folates occur mainly as one-carbon substituted derivatives of THF and are predominantly polyglutamylated compounds containing up to 8 γ-glutamyl residues. (Click image to enlarge.)

The precursors of THF are synthesized in the cytosol and plastids
The biosynthetic steps towards THF in bacteria, fungi and plants are nearly the same. All the genes and enzymes specific to the pathway have been characterized in plants, at least in Arabidopsis thaliana (Ravanel et al. 2004; Rébeillé et al. 2006). The pterin and PABA moieties are first synthesized in separate branches. The synthesis of dihydropterin from GTP involves the enzymes GTP-cyclohydrolase I, dihydroneopterintriphosphate pyrophosphatase and dihydroneopterin aldolase (reactions 1-3, Figure 2). Genomic evidence suggests that the three enzymes of the pterin branch are present in the cytosol. PABA is synthesized from chorismate, a product of the shikimate pathway, through the reactions catalyzed by aminodeoxychorismate synthase and aminodeoxychorismate lyase. These reactions take place in the stroma of chloroplasts (reactions 4-5, Figure 2). The recent development of engineering strategies to increase folates content in transgenic tomato plants have revealed that both the synthesis of the pterin and PABA moieties are limiting factors for THF synthesis, at least in fruits and seeds (Diaz de la Garza et al. 2004; Storozhenko et al. 2007).

Figure 2 The tetrahydrofolate biosynthetic pathway in plants. The enzymes involved in the synthesis of THF are 1, GTP cyclohydrolase I; 2, dihydroneopterintriphosphate pyrophosphatase; 3, dihydroneopterin aldolase;4, aminodeoxychorismate (ADC) synthase; 5, ADC lyase; 6, hydroxymethyldihydropterin pyrophosphokinase; 7, dihydropteroate synthase; 8, dihydrofolate synthetase; 9, dihydrofolate reductase; 10, folylpolyglutamate synthetase. Probable transport steps for THF and precursors are indicated by dotted arrows. THF-Glu_n, THF with n glutamates; Glu, glutamate. (Click image to enlarge.)

THF is synthesized in mitochondria
The last five steps of the pathway leading to the assembly of dihydropterin, PABA and glutamate are located in mitochondria (Figure 2). The first two reactions are catalyzed by a bifunctional hydroxymethyldihydropterin pyrophosphokinase / dihydropteroate synthase enzyme (reactions 6-7, Figure 2). The dihydropteroate synthase activity is a potential regulatory point of the folate biosynthetic pathway because the enzyme is feedback inhibited by dihydropteroate, product of the reaction, dihydrofolate, and THF monoglutamate. Also, the structural analogs of PABA (sulfonamides) inhibit strongly the plant dihydropteroate synthase and behave as herbicides. Dihydrofolate synthetase, which catalyzes the attachment of the first glutamate residue of THF (reaction 8, Figure 2), is also essential for plant development because a knockout in the corresponding gene is embryo-lethal in Arabidopsis. After the first glutamylation step, dihydrofolate is reduced to THF by the enzyme dihydrofolate reductase (reaction 9, Figure 2). Higher plants and protozoa possess a bifunctional enzyme bearing dihydrofolate reductase and thymidylate synthase activities. Thymidylate synthase catalyzes the methylation of dUMP into dTMP (for DNA synthesis) in the presence of CH₂-THF, a reaction that is unique because CH₂-THF acts both as a C1-unit donor and a reducing agent, thus producing dihydrofolate. Several lines of evidence suggest that plant cells possess dihydrofolate reductase / thymidylate synthase isoforms in the mitochondria, the plastids, and the cytosol.

Intracellular traffic of folate-coenzymes
The last step of the plant THF synthesis pathway is the sequential addition of γ-linked glutamate residues to THF, a reaction catalyzed by folylpolyglutamate synthetase (reaction 10, Figure 2). In all eukaryotes studied so far, folylpolyglutamate synthetase isoforms were found in each subcellular compartment containing folylpolyglutamates, suggesting that these derivatives cannot cross membranes and must be synthesized in situ. Therefore, it is not surprising that in Arabidopsis folylpolyglutamate synthetase is present as three distinct isoforms located in the mitochondria, the cytosol, and the chloroplasts, respectively. In Arabidopsis, each folylpolyglutamate synthetase isoform is encoded by a separate gene, a situation that is unique among eukaryotes. As shown in Figure 2, the pathway for THF synthesis in plants is split between plastids, mitochondria and cytosol. This complex distribution, which is not observed in others eukaryotes, suggests a sophisticated traffic of folate-coenzymes and their biosynthetic intermediates between the organelles via the cytosol. The only plant folate transporters identified so far are located in the chloroplast envelope (Rébeillé et al. 2006). Among these compartments, mitochondria occupy a central position because they have to take up the pterin and PABA units from the cytosol and are the only site for de novo synthesis of the fully active THF molecule.

Importance of Mitochondria in Biotin Synthesis

Biotin synthesis occurs in 4 steps
In plants, as well as in all known microbes, biotin is synthesized from pimeloyl-CoA and alanine through four enzymatic steps comprising 7-keto-8-aminopelargonic acid synthase (KAPA synthase), 7,8-diaminopelargonic acid (DAPA) aminotransferase, dethiobiotin synthase, and biotin synthase (Figure 3). Initial information on biotin synthesis and transport in higher plants came from analysis of the bio1 biotin auxotroph of Arabidopsis thaliana. Seeds homozygous for the mutation failed to develop unless exogenous biotin, dethiobiotin or DAPA was supplied to the plant. The E. coli bioA gene, which codes for DAPA aminotransferase, could genetically complement the bio1 mutation, demonstrating that bio1/bio1 mutant plants are defective in this enzyme. Treatment of a biotin-overexpressing strain of lavender cells with 3H-pimelic acid showed that all the intermediates of biotin synthesis established in bacteria plus the novel metabolite 9-mercaptodethiobiotin (9-mDTB) accumulate in plants. This demonstrated that the pathway of biotin synthesis in bacteria is conserved in plants and that the reaction catalyzed by biotin synthase proceeds in two distinct steps involving 9-mDTB as an intermediate (Alban et al. 2000).

Figure 3 The biotin biosynthetic pathway in plants. A: The biotin cofactor. Biotin is covalently attached to the apoenzyme by an amide linkage between the carboxyl group of its valerate lateral chain and a lysine of the protein. The carboxybiotinyl-enzyme is formed in presence of a bicarbonate ion through an ATP depend reaction catalyzed by the biotin carboxylase. B: The four steps involved in biotin synthesis. 1, KAPA synthase (BIO4); 2, DAPA aminotransferase-BIO1/dethiobiotin synthase-BIO3 (BIO3-BIO1); 3, biotin synthase (BIO2). KAPA, 7-keto-8-aminopelargonic acid; DAPA, 7,8-diaminopelargonic acid; DTB, dethiobiotin. The putative translocators involved in the traffic of biotin and precursors between mitochondria and cytosol are shown. (Click image to enlarge.)

The last step of biotin synthesis involves a protein complex located in mitochondria
The catalytic mechanism of the last step of biotin biosynthesis is still unclear. In E. coli the conversion of dethiobiotin to biotin is catalyzed by a complex involving at least 3 proteins in addition to biotin synthase which alone is not able to support this reaction (Marquet et al. 2001). A cDNA coding Arabidopsis biotin synthase (named Bio2) was cloned by functional complementation of an E. coli bioB biotin auxotroph mutant (Baldet and Ruffet 1996) and the protein was shown to be located in mitochondria (Baldet et al. 1997). The recent development of radiochemical and biological detection methods allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein (Picciocchi et al. 2001). As its bacterial counterparts, purified Bio2 protein is a homodimer that, in its active form, coordinates a [2Fe-2S] and a [4Fe-4S] cluster per monomer. Also, the purified enzyme alone is not able to support biotin synthesis. Heterologous interactions between the plant Bio2 protein and bacterial accessory proteins yield a functional biotin synthase complex. Combination experiments using purified Bio2 protein and extracts from pea leaf or potato tuber organelles showed that only mitochondrial fractions could elicit biotin formation in the plant reconstituted system confirming the mitochondrial location of biotin synthesis in plants (Picciocchi et al. 2001).

A biochemical screening of potato mitochondrial matrix (fractionation and in vitro reconstitution experiments) together with a genomic based search in the A. thaliana databank allowed us to identify mitochondrial adrenodoxin (Adx), adrenodoxin reductase (AdxR) and cysteine desulfurase (Nfs1) proteins as essential components for the plant biotin synthase reaction (Picciocchi et al. 2003). The in vitro stimulation of biotin synthase activity by the Nfs1 protein strongly supports the idea that cysteine is the initial sulphur donor for biotin in plant mitochondria. The purification of recombinant Adx and AdxR of A. thaliana enable us to establish the first biochemical characterization of a plant Adx/AdxR reaction. These two purified recombinant proteins formed in vitro an efficient low potential electron transfer chain that interacted with the bio2 gene product to reconstitute a functional plant biotin synthase complex. The role of this physiological reduction system is the reductive cleavage of S-adenosylmethionine (AdoMet), an obligatory cofactor of the reaction, through the [4Fe-4S] centre of the enzyme. Thus, Bio2 from Arabidopsis is the first identified protein partner in higher plants for this specific mitochondrial redox chain.

In addition to their implication in biotin synthase reaction, mitochondrial Adx/AdxR redox system and Nfs1 protein could be also involved in the synthesis of lipoate cofactor which also occurs in plant mitochondria (Douce et al. 2001), the lipoate synthase and biotin synthase reactions being mechanistically related. Also, the yeast proteins homologous to Adx, AdxR and Nfs1 (namely Yah1p, Arh1p and Nfs1p) have been recently identified as key components of iron-sulphur cluster assembly machinery. Consequently, these proteins could have a dual function, a specific function in the biotin synthase and lipoate synthase reactions and a more general role in biosynthesis of Fe-S clusters for other redox enzymes, e.g. aconitase.

Intracellular traffic of biotin and its precursors
Finally, the mitochondrial location of the biotin synthase reaction raises new interesting questions. Indeed, synthesis of biotin depends on the presence of dethiobiotin and AdoMet in plant mitochondria. Dethiobiotin is synthesized from 7-keto-8-amino pelargonic acid (KAPA) by a bifunctional enzyme catalyzing both DAPA aminotransferase and dethiobiotin synthase reactions. In Arabidopsis this enzyme is encoded by a chimeric locus (BIO3-BIO1) resulting from a gene fusion event that occurred early in the evolution of eukaryotes (Muralla et al. 2008). This original enzyme, putatively localized in the mitochondrial matrix (Figure 3) has not been yet characterized. Interestingly, KAPA, the substrate of this complex reaction is produced in the cytosol (Pinon et al. 2005). In plant cells, AdoMet is synthesized in the cytosol, a compartment in which free biotin also accumulates. Specific carriers (that remain to be identified) are thus required to ensure the import of AdoMet into the mitochondrial matrix on one hand and to export biotin to the cytosol on the other hand (Figure 3). Furthermore, the control of AdoMet and KAPA fluxes from cytosol to mitochondrial matrix constitutes a potential additional regulatory level of biotin synthase activity in plant mitochondria.

In conclusion, the biochemical characterization of the A. thaliana biotin synthase reaction demonstrates the importance of plant mitochondria in biotin biosynthesis. Moreover, the identification and the involvement of mitochondrial Adx/AdxR redox chain in this vitamin synthesis opens up future prospects in the understanding of the biotin synthase activity regulation in plant cells.

Figure 4 The various functions (demonstrated or putative) associated with the adrenodoxin/adrenodoxin reductase redox system and Nfs1 protein in plant mitochondria. The Adx/AdxR redox system from the matrix space is probably the physiological electron donor for numerous metabolic reactions. Our work demonstrates the implication of these proteins in biotin synthase reaction. Mitochondrial lipoate synthase (Lip1) is believed to have a similar reaction mechanism. In plant mitochondria an iron-sulfur assembly machinery similar to that described in yeast has been identified. Thus, the intervention of the Adx/AdxR redox system and of Nfs1 protein from Arabidopsis in biosynthesis of Fe-S clusters for mitochondrial proteins (including bio2 and Lip1) and cytosolic proteins is also possible. Finally, the participation of this matrix redox system in other, still unidentified, cell functions cannot be excluded. DTB, dethiobiotin. (Click image to enlarge.)

References

Alban, C., Job, D. and Douce, R. (2000) Biotin metabolism in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 17–47.

Baldet, P. and Ruffet, M. L. (1996) Biotin synthesis in higher plants: isolation of a cDNA encoding Arabidopsis thaliana bioB–gene product equivalent by functional complementation of a biotin auxotroph mutant bioB105 of Escherichia coli K12. C.R. Acad. Sci. Paris 309: 99–106.

Baldet, P., Alban, C. and Douce, R. (1997) Biotin synthesis in higher plants: purification of bio B gene product equivalent from Arabidopsis thaliana overexpressed in Escherichia coli and its subcellular localization in pea leaf cells. FEBS Lett. 419: 206–210.

Diaz de la Garza, R., Quinlivan, E. P., Klaus, S. M., Basset, G. J., Gregory, J. F., 3rd and Hanson, A. D. (2004) Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis. Proc. Natl Acad. Sci. U S A 101: 13720–13725.

Hanson, A. D. and Roje, S. (2001) One–carbon metabolism in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 119–137.

Marquet, A., Tse Sum Bui, B. and Florentin, D. (2001) Biosynthesis of biotin and lipoic acid. Vitam. Horm. 61: 51–101.

Muralla, R., Chen, E., Sweeney, C., Gray, J. A., Dickerman, A., Nikolau, B. J. and Meinke, D. (2008) A bifunctional Locus (BIO3–BIO1) required for biotin synthesis in Arabidopsis. Plant Physiol. 146: 60–73.

Picciocchi, A., Douce, R. and Alban, C. (2001) Biochemical characterization of the Arabidopsis thaliana biotin synthase reaction. The importance of mitochondria in biotin synthesis. Plant Physiol. 127: 1224–1233.

Picciocchi, A., Douce, R., and Alban. C. (2003) The plant biotin synthase reaction: identification and characterization of essential mitochondrial accessory protein components. J. Biol. Chem. 278: 24966–24975.

Pinon, V., Ravanel, S, Douce, R. and Alban C. (2005) Biotin synthesis in plants. The first committed step of the pathway is catalyzed by a cytosolic 7–keto–8–aminopelargonic acid synthase. Plant Physiol. 139: 1666–1676.

Ravanel, S., Douce, R. and Rébeillé, F. (2004) The uniqueness of tetrahydrofolate synthesis and one–carbon metabolism in higher plants. In: Advances in Photosynthesis and Respiration. Plant mitochondria, from Genome to Function, eds. D. A. Day, A. H. Millar and J. Whelan, 17: 277–292.

Rébeillé F, Ravanel S, Jabrin S, Douce R, Storozhenko S, Van Der Straeten D (2006) Folates in plants: biosynthesis, distribution, and enhancement. Physiol Plant 126: 330–342.

Scott, J., Rébeillé, F. and Fletcher, J. (2000) Folic acid and folates: the feasibility for nutritional enhancement in plant foods. J. Sci. Food Agric. 80: 795–824.

Storozhenko S, De Brouwer V, Volckaert M, Navarrete O, Blancquaert D, Zhang GF, Lambert W, Van Der Straeten D (2007) Folate fortification of rice by metabolic engineering. Nat Biotechnol 25: 1277–1279.