View Article Online / Journal Homepage / Table of Contents for this issue

REVIEW www.rsc.org/npr | Natural Product Reports Roles of vitamins B5, B8, B9, B12 and molybdenum cofactor at cellular and organismal levels†

Fabrice Rebeill´ e,*´ a Stephane´ Ravanel,a Andree´ Marquet,b Ralf R. Mendel,c Alison G. Smithd and Martin J. Warrene

Received (in Cambridge, UK) 14th June 2007 First published as an Advance Article on the web 20th August 2007 DOI: 10.1039/b703104c

Covering: 1984 to 2007

Many efforts have been made in recent decades to understand how coenzymes, including vitamins, are synthesised in organisms. In the present review, we describe the most recent findings about the biological roles of five coenzymes: folate (vitamin B9), pantothenate (vitamin B5), cobalamin (vitamin B12), biotin (vitamin B8) and molybdenum cofactor (Moco). In the first part, we will emphasise their biological functions, including the specific roles found in some organisms. In the second part we will present some nutritional aspects and potential strategies to enhance the cofactor contents in organisms of interest.

1 Introduction 1 Introduction 2 Biological functions 2.1 Main functions found in all organisms Cofactors are small molecules (at least compared to the size 2.1.1 Nucleic acid synthesis: the role of folate of a protein) that facilitate an enzyme to catalyze a reaction. 2.1.2 The methylation cycle: the roles of folate and These ‘chemical tools’ can be inorganic (metal ions or clusters) or cobalamin organic (coenzymes) and are generally involved in group transfer 2.1.3 Fatty acid biosynthesis and gluconeogenesis: the roles or redox reactions. They can act as co-substrates or be permanently of biotin and pantothenate associated with the structure of the enzyme (prosthetic groups). 2.1.4 Redox reactions: the role of Moco A large number of these coenzymes are derived from vitamins. 2.1.5 Other metabolic functions for folate, biotin and Vitamins, by definition, are dietary substances required for good cobalamin health and normal development of animals. Most of them are only 2.2 The main differences among eukaryotic organisms synthesised by microorganisms and plants. During the course of Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. 2.2.1 Compartmentalisation animal evolution, the ability to biosynthesise these compounds 2.2.2 Specific needs in some eukaryotes has been lost and, instead, elaborate uptake mechanisms have 3 Nutritional aspects been developed. As many vitamins are only required in trace 3.1 Effects of deficiency on human health quantities, their biosynthesis is normally strictly controlled and the 3.2 Main dietary sources enzymes involved are produced in vanishingly small amounts. This 3.3 Strategies for enhancement is why it has been extremely difficult to elucidate their complete 4 Conclusion: compartmentalisation, a challenging biosynthetic pathways, and it still remains the case that many steps area within the biosynthesis of vitamins are poorly understood (see the 5 References review by Webb and Smith in this issue). Because they are essential in all organisms and are required in a number of biological processes, vitamins are of considerable interest in terms of what they do and how they are made. In aLaboratoire de Physiologie Cellulaire Veg´ etale,´ UMR5168, Universite´ Joseph Fourier-CNRS-CEA-INRA, Institut de Recherche en Technologies et the post-genomic era there now exist opportunities to understand Sciences du Vivant, CEA-Grenoble, 17 rue des Martyrs, F-38054, Grenoble, fully how these compounds are synthesised and what their whole Cedex 9, France. E-mail: [email protected]; Fax: +33 438-78-50-91; Tel: +33 cellular functions are. These functions can be quite complex 438-78-44-93 because one particular vitamin may have various metabolic bDepartment of Chemistry, Universite´ Pierre et Marie Curie, UMR CNRS 7613, 75252, Paris, France. E-mail: [email protected] and chemical roles. In addition, this role may fluctuate from cDepartment of Plant Biology, Technical University of Braunschweig, 38106, one organism to another depending on the presence of specific Braunschweig, Germany. E-mail: [email protected] metabolisms (for example photosynthesis in plants). Increasing dDepartment of Plant Sciences, University of Cambridge, Cambridge, CB2 our knowledge concerning their synthesis and function is a prereq- 3EA, UK. E-mail: [email protected] uisite to develop new strategies for health and/or wealth creation, eDepartment of Biochemistry, University of Kent, Canterbury, UK. E-mail: [email protected] including improvement of food quality, design of new antibiotics † This paper was published as part of a themed issue on vitamins and targeting vitamin biosynthesis, and engineering synthesis of new cofactors. compounds.

This journal is © The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 949–962 | 949 View Article Online

Dr Fabrice Rebeill´ e´ is Director of Research at the Commissariat a` l’Energie Atomique (CEA), Grenoble, France. He graduated from the University of Grenoble where he obtained a Diploma in Pharmacology in 1978 and a PhD in 1983. After a post-doc in Prof. M. D. Hatch’s lab in CSIRO, Canberra, he made a career within the CEA and was also Professor of Biochemistry at the University of Grenoble for four years. His main research activities are focused on plant metabolism, including phosphate metabolism, photosynthesis, photorespiration, and more

recently, folate biosynthesis and C1 metabolism.

Dr Stephane´ Ravanel is Associate Professor at the University of Grenoble, France. He studied biochemistry and molecular biology at the universities of Lyon and Grenoble, and then completed a PhD in plant biology in 1995. After a post-doc in Prof. Rochaix’s lab at the University of Geneva, he joined the University of Grenoble and the lab of Plant Cell Physiology in 1998. His present research focuses on the molecular, biochemical and regulatory aspects of folate and one-carbon metabolism in plants.

Stephane´ Ravanel Fabrice Rebeill´ e´

Here, we will describe, as examples, the role of five coenzymes: folate (vitamin B9), pantothenate (vitamin B5), cobalamin (vita- min B12), biotin (vitamin B8) and molybdenum cofactor (Moco). Firstly, their biological functions will be emphasised, including the specific roles found in some organisms. In the second part we will present some nutritional aspects concerning these coenzymes and potential strategies to enhance the cofactor contents in organisms of interest.

2 Biological functions

2.1 Main functions found in all organisms Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. Cofactors are required in almost all important metabolic path- ways. Because they are specialised in certain types of reaction, one particular cofactor can be involved in several pathways and, conversely, several cofactors can be required in one particular pathway. The following section considers the main areas of metabolism in which the above five coenzymes are involved.

2.1.1 Nucleic acid synthesis: the role of folate. The syntheses of both purine and pyrimidine nucleotides require a folate

coenzyme. Folates are involved in ‘one-carbon’ unit (C1 unit) Fig. 1 Chemical structure of THF and major reactions of C1 metabolism.

transfer reactions, also called ‘C1 metabolism’. Folate is a generic THF is substituted at the N-5 and/or N-10 positions by C1 units having term that represents a family of molecules (Fig. 1) deriving from various oxidation states. There are generally between 4 and 8 glutamate

tetrahydrofolate (5,6,7,8-tetrahydropteroylpolyglutamate, THF). residues. Serine and, to a lesser extent, formate, are the sources of C1 units.

Chemically, these folate molecules are composed of a pterin ring, The highest flux of C1 units occurs through methionine synthesis to sustain a p-aminobenzoic acid (pABA) unit and a polyglutamate chain AdoMet turnover and all the methylation reactions, which explains why with a variable number (1 to 14) of glutamate residues.1 Their 5-methyltetrahydrofolate is the dominant folate species.

role is to transport and donate C1 units. The C1 units transported by the vitamin arise essentially from the reaction catalyzed by

serine hydroxymethyltransferase (SHMT) that converts serine into on the nature of the C1 unit carried, the folate coenzyme will be glycine.2 Formate is also a potential, although minor, source of involved in various pathways (summarised in Fig. 1). Thus, the 3 C1 units. Once attached to the THF body, these C1 units can folate pool is a complex mixture of related molecules differing be reduced or oxidised, from methyl (the most reduced), via in the oxidation state of the pterin ring (di- or tetrahydrofolate), 4 methylene, to formyl or methenyl (the most oxidised). Depending in the oxidation state of the C1 unit carried and in the length of

950 | Nat. Prod. Rep., 2007, 24, 949–962 This journal is © The Royal Society of Chemistry 2007 View Article Online

the glutamate chain. Only the most reduced form of the cofactor involved in the de novo synthesis of THF in folate-autotroph 6 (tetrahydrofolate) can transport these C1 units. organisms. Synthesis of the purine ring provides AMP and GMP bases for DNA and RNA strands, as well as for coenzymes such as 2.1.2 The methylation cycle: the roles of folate and cobalamin. NAD(P)+, FAD, CoA and S-adenosylmethionine (AdoMet). In Methionine (Met) is an essential amino acid not only required plants, these nucleotides are also precursors for purine alkaloids for the synthesis of protein but also for the formation of S- and the cytokinin hormones. The pathway for their synthesis is adenosylmethionine (AdoMet), the universal methyl donor and 7,8 similar in plants, animals and micro-organisms.5 It is a complex a key element in the ‘methylation cycle’ (Fig. 3a). In fact, process requiring 13 steps from ribose 5-phosphate. The fourth and 80% of the free Met present in the cell is used in this cycle, tenth steps, respectively catalyzed by glycinamide ribonucleotide whose function is continuously to supply with AdoMet the transformylase and aminoimidazole carboxamide ribonucleotide dozens of methyltransferase enzymes present in all cells. These methyltransferases, as the name suggests, transfer the methyl transformylase, involve the addition of a C1 unit. This C1 unit is provided by the 10-formyltetrahydrofolate derivative (Fig. 2a), group from AdoMet to a very large range of substrates for the inserting the carbons which become C-2 and C-8 of the purine synthesis of numerous compounds, including chlorophyll, lipids, 4 ring. lignin, hormones and vitamins. They are also involved in a wide range of functions such as regulation of gene expression (methylation of DNA and histones)9,10 or regulation and repair of proteins (rubisco enzyme, myelin basic protein etc.)11,12 The central step in this cycle is the methylation of homocysteine (Hcy), a reaction catalyzed by methionine synthase and responsible for the continual regeneration of Met (Fig. 3a). In this reaction, the 13 C1 unit transferred to Hcy arises from 5-methyltetrahydrofolate, making this folate derivative the de facto source of all methyl groups. Interestingly, there are two types of methionine synthase whose activities depend or not on the presence of a cobalamin cofactor. The cobalamin-independent form of the enzyme (MetE) is found in plants and fungi, the cobalamin-dependent type (MetH) is found in animals14 (see below), and both types exist in enterobacteria and certain algae. Thus, this second class of enzyme (MetH) requires two coenzymes: folate and cobalamin. Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55.

Fig. 2 Role of folates in nucleotide synthesis. A: reactions involved in purine synthesis: 1) GAR transformylase; 2) AICAR transformylase. THF generated during these reactions is recycled back to 10-CHO-THF or

5,10-CH2-THF, as shown in Fig. 1. B: reactions involved in thymidylate

synthesis: 3) thymidylate synthase; in this reaction, 5,10-CH2-THF is

both a C1 unit donor and a reducing agent; 4) dihydrofolate reductase;

5) SHMT. In each case the C1 unit added is indicated by a box. GAR, glycinamide ribonucleotide; FGAR, formyl glycinamide ribonucleotide; AICAR, aminoimidazole carboxamide ribonucleotide; FAICAR, formyl aminoimidazole carboxamide ribonucleotide; 10-CHO-THF, 10-formyl-

tetrahydrofolate; 5,10-CH2-THF, 5,10-methylenetetrahydrofolate.

The synthesis of thymidylate is also closely linked to C1 metabolism. Indeed, the enzyme that converts the uracil base into Fig. 3 The methylation cycle. A, overview of the methylation cycle; 1) methionine synthase (either MetE or MetH); 2) AdoMet synthetase; the thymine base, to form the pyrimidine (dTMP) found uniquely 3) methyl transferases; 4) S-adenosylhomocysteine hydrolase; 5) SHMT; in DNA, uses the folate vitamin with the C1 unit attached as 5,10- 6) methylenetetrahydrofolate reductase; serine is the source of the C1 units methylenetetrahydrofolate (Fig. 2b). Whereas THF is regenerated required to methylate X; 5,10-CH2-THF, 5,10-methylenetetrahydrofolate;

during purine synthesis, it is dihydrofolate (DHF) that is formed 5-CH3-THF, 5-methyltetrahydrofolate; AdoHcy, S-adenosylhomocys- during dTMP synthesis. The latter is converted back to THF by teine. B, the intermediary role of cobalamin in the B12-dependent the important enzyme dihydrofolate reductase, an enzyme also methionine synthase (MetH).

This journal is © The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 949–962 | 951 View Article Online

The B12-dependent methionine synthase (MetH) catalyses the transfer of a methyl group from methyltetrahydrofolate to Hcy.15 In essence, this is the same reaction as catalysed by the B12- independent enzyme (MetE), and both enzymes face the same challenge of transferring the methyl group from a very poor leaving group, the tertiary amine of methyltetrahydrofolate, to the sulfur of Hcy.16 The B12-dependent enzyme differs from the independent enzyme in that it uses cobalamin as an intermediary methyl group carrier (Fig. 3b), whereas the B12-independent enzyme catalyses direct transfer of the methyl group. The employment of cobalamin appears to make the process much more favourable, as the B12 enzyme has significantly a higher (by two orders of magnitude) 17 kcat value. Methionine synthase (MetH) is a large modular enzyme consisting of 4 distinct regions representing (i) Hcy-binding, (ii) methyltetrahydrofolate-binding, (iii) cobalamin-binding and (iv) AdoMet-binding domains.15 The structure of the full length protein has not been determined but the topology of the individual domains has been elucidated and this, coupled with a large amount of mechanistic work, has allowed a comprehensive overview of the catalytic cycle of the enzyme to be resolved. The enzyme operates by binding Hcy to a specific site in domain (i) that is defined, in part, by the presence of an essential zinc ion, itself ligated to the region via three cysteine residues. Methyl cobalamin is formed by the transfer of a methyl group from methyltetrahydrofolate bound to domain (ii) to Co(I) cobalamin bound in domain (iii). This transfer is facilitated by the strong nucleophilicity of the Co(I) form of cobalamin. Domain (iii) housing the Co(III) methylcobalamin then interacts with the Hcy binding domain (i) to generate methionine and Co(I) cobalamin. To facilitate the passage and transfer of the methyl group between the various substitutents of the catalytic cycle, the enzyme has to undergo large-scale conformational changes.18 Moreover, in the presence of oxygen, approximately once every 200 turnovers, the cobalt ion in cobalamin becomes oxidised to the Co(II) form, which is

Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. catalytically inactive. The enzyme is able to reactivate itself through its AdoMet binding domain (iv). Through an interaction with a flavoprotein, which in E. coli is flavodoxin and in humans is methionine synthase reductase, the cobalt ion is reduced back to the Co(I) form. This is rapidly methylated by AdoMet to generate methylcobalamin and allows the catalytic cycle of the enzyme to be restored.15

2.1.3 Fatty acid biosynthesis and gluconeogenesis: the roles of biotin and pantothenate. Fatty acid biosynthesis is a repeated series of reactions that incorporate acetyl moieties (two-carbon units) of acetyl-CoA into an acyl group to form a 16- or 18- carbon-long chain. Both biotin and pantothenate are essential as cofactors for the enzymes involved in this process (Fig. 4), acetyl- CoA carboxylase (ACCase), a biotin-dependent enzyme, and fatty acid synthase (FAS). FAS catalyzes a set of repetitive reactions including condensation of the two-carbon units with the growing Fig. 4 The role of pantothenate and biotin in fatty acid synthesis. A, Schematic representation of the fatty acid synthesis pathway showing fatty acyl chain, then reduction, dehydration and reduction again where the two vitamins are required; B, reaction catalyzed by the to obtain a fully reduced acyl group. FAS is a complex system acetyl-CoA carboxylase (ACCase) showing the role of biotin in the working either as a multifunctional enzyme characterised by large − ATP-dependent activation of CO2 (in the form of HCO3 ) and its subunits (animal, yeast) or as individual proteins functioning transfer to acetyl-CoA; C, the structure of CoA and ACP showing the much like a metabolic pathway (plants, most bacteria). In both pantothenate unit (boxed) of the molecules. cases, the FAS system requires an essential protein cofactor: the acyl-carrier protein (ACP), though in the multifunctional

952 | Nat. Prod. Rep., 2007, 24, 949–962 This journal is © The Royal Society of Chemistry 2007 View Article Online

enzyme the ACP is incorporated into the enzyme as one of its metal is complexed by a unique tricyclic pterin, molybdopterin,23 domains. to produce the molybdenum cofactor, and only in this form ACP is a small protein of about 80 amino acids to which is is it inserted into its diverse target proteins. During catalysis, bound a 4-phosphopantetheine moiety, derived from pantothen- it shuttles between oxidation state +4 and +6, and for several ate (Fig. 4c). This group is also found in coenzyme A (CoA), and enzymes the reaction mechanism has been worked out in detail.24 one role of the cofactor is to solubilise hydrophobic acyl groups. The task of the pterin moiety, however, is less defined. Clearly, Moreover, the thioester linkage between the thiol group of 4- the pterin scaffold has to position the catalytic metal correctly phosphopantetheine and the acyl group provides a good leaving within the active site, thereby controlling its redox behaviour. It group for the formation of C–C bonds. As well as its role in fatty has also become clear that the pterin moiety participates with acid synthesis, CoA plays an important role in key metabolic its ring system in the electron transfer to or from the Mo atom. reactions such as the TCA cycle and secondary metabolism Yet, it appears that the cofactor does not participate directly in including lignin, flavonoid and terpenoid biosynthesis. In E. coli, catalysis. Obviously, it plays a more indirect role in modulating the 4% of the known enzymes have been shown to require CoA.19 4- reduction potential and reactivity of the molybdenum center. The Phosphopantetheine is also the prosthetic group of polyketide and pterin, with its several possible reduction states as well as different non-ribosomal peptide synthases,20 involved in the biosynthesis of structural conformations, could also be important for channelling many antibiotics, toxins and pigments. electrons to other prosthetic groups. Biotin on the other hand has a much narrower role as the cofactor for a limited number of enzymes in central metabolism (see below for other functions). In all cases it is covalently

bound to its partner enzymes, and serves as a CO2 carrier between bicarbonate and the acceptor substrate (Fig. 4b). N- − Carboxybiotin is first produced from HCO3 and ATP, and this

activated form of CO2 is then transferred to the substrate, in this case acetyl-CoA, to produce malonyl-CoA, the elongating unit. Beside ACCase, there are three other metabolically important car- 2.1.5 Other metabolic functions for folate, biotin and cobal- 21 boxylases already widely reviewed. The first one is the pyruvate amin. Beside their role in C1 metabolism, folate derivatives act carboxylase, which catalyses an important step in gluconeogenesis also as chromophores. This property arises from the aromatic by converting pyruvate into oxaloacetate. This reaction also serves nature of the pterin and p-aminobenzoyl rings. This function is to replenish the oxaloacetate pool available for the tricarboxylic exploited by a certain class of enzymes named photolyases and acid cycle. The second one is the propionyl-CoA carboxylase involved in DNA repair following UV-B light damage.25,26 UV-B that allows propionate (arising from the catabolism of odd-chain light (280–310 nm) induces covalent bonds between two adjacent fatty acids and branched-chain amino acids) to enter the citric pyrimidines. These pyrimidine dimers have deleterious effects, acid cycle, by converting propionyl-CoA into methylmalonyl- including inhibition of replication and transcription, followed CoA. Methylmalonyl-CoA is then transformed into succinate by growth arrest and cell death. The removal of these covalent by two further steps. Finally, 3-methylcrotonyl-CoA carboxylase bonds is catalyzed by photolyases, a class of enzymes using the

Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. converts 3-methylcrotonyl-CoA, an intermediate in the catabolism energy of UV-A/blue light (350–600 nm), and two cofactors: a of leucine, into 3-methylglutaconyl-CoA. This molecule eventually 5,10-methenyltetrahydrofolate and a flavin cofactor FAD, derived results in acetoacetate and acetyl-CoA formation. from riboflavin (vitamin B2). The folate chromophore functions as a photoantenna which absorbs the light energy and transfers 2.1.4 Redox reactions: the role of Moco. Oxido-reduction the resultant excitation to the FAD cofactor that, in turn, breaks reactions are of ultimate importance in cell biology because most the undesired covalent bond.25,26 of the free-energy available in living organisms relies on this The search for other functions of biotin is presently a very type of reaction. As a matter of fact, a very large variety of active field.27 Early studies had shown that biotin influences the substrates are either reduced or oxidised in almost all aspects expression of some genes,28 and now more than 2000 human of cellular metabolism. These reactions involve various cofactors genes that depend on biotin have been identified. This effect such as metal ions, iron–sulfur clusters, hemes, glutathiones, is mediated by different cell signals or transcription factors: nicotinamides (from vitamin B3, also called niacin or vitamin biotinyl-AMP, NF-jB, Sp1 and Sp3, receptor tyrosine kinases. PP), flavins (from riboflavin, vitamin B2), ascorbate (vitamin The best documented area is the biotinylation of histones. It is C) or Moco. It is not possible to describe here the role of all now established that biotin is covalently bound to histones through these molecules, and we will concentrate on the molybdenum amide bonds with distinct lysine residues. This post-translational cofactor (Moco), to which less attention has been paid up to modification, regulated by the biotin level, influences the structure now. There are more than 50 molybdenum-containing enzymes of chromatin, and hence gene expression. The biotinylation of known, most of them of bacterial origin, that participate in histones appears to play a role in cell proliferation, gene silencing, essential redox reactions in the global nitrogen-cycle (involving and the cellular response to DNA repair. This reaction is catalyzed the molybdenum enzymes nitrogenase, nitrate reductase, nitrite by biotinidase and holocarboxylase synthetase, as illustrated in oxidase), the sulfur-cycle (involving sulfite oxidase and DMSO- Fig. 6. reductase) and the carbon-cycle (involving CO-dehydrogenase, Vitamin B12- or cobalamin-dependent enzymes are required in aldehyde oxidases and formate dehydrogenase).22 With the excep- three broad classes of reactions: (i) B12-dependent isomerases, tion of bacterial nitrogenase, molybdenum as a catalytically active (ii) B12-dependent methyltransferases and (iii) B12-dependent

This journal is © The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 949–962 | 953 View Article Online

reductive dehalogenases.29 In the B12-dependent isomerases, the in the cytosol where the methylation cycle is also exclusively

biological form of cobalamin is adenosylcobalamin, the coen- located. Thus, most C1 units produced in the mitochondria are zyme form of B12.30 Here, the properties of the weak cobalt– exported to the cytosol to sustain these activities. In plants, the carbon bond are exploited through homolytic cleavage, generating situation appears more complex for several reasons. Firstly, plants a5-deoxyadeonsyl radical and cob(II)alamin. The radical is have plastids that also contain SHMT, the enzyme involved in 4 used to promote a variety of complex 1,2-rearrangements that the generation of C1 units. Secondly, whereas the methylation are largely associated with anaerobic fermentative processes. cycle appears to be exclusively located in the cytosol, as it is in These include the diol dehydratases of propanediol and glycerol other organisms, nucleotide synthesis in plants is also located in metabolism, ethanolamine ammonia lyase and the amino mutases organelles: purines are essentially synthesised in the plastids,36 that function in the fermentation of glutamate, lysine, leucine whereas dTMP is produced in the mitochondria, the plastids and and ornithine.31 This class also includes the reactions associated possibly the cytosol, as suggested by the presence of thymidylate with the methylmalonyl-CoA mutase and the B12-dependent synthase in these compartments.33 ribonucleotide reductase. There appear to be two subclasses of b) Biotin. Biotin synthesis in plants and bacteria appears to isomerases that differ fundamentally on how the coenzyme binds follow a similar pathway from pimeloyl-CoA37 (see review by to the enzyme. In class I enzymes, the bond between the lower base Webb and Smith in this issue), but in plants the first three of B12, the dimethylbenzimidazole (DMB) group and the cobalt enzymes are cytosolic and the terminal enzyme, biotin synthase, ion is replaced by a link with an imidazole side chain of a specific is mitochondrial. The Arabidopsis BIO2 gene encoding one of the histidine residue, and thus the coenzyme is said to bind in a base- subunits of this enzyme has sequence similarity with the bacterial off conformation. In class II enzymes, the DMB link is retained, enzyme, but appears to encode an N-terminal extension not and the coenzyme is said to bind in a base-on conformation.29 present in the latter. This extra sequence acts as a mitochondrial Methylcobalamin is associated with methylation reactions, as targeting peptide. Interestingly, when a truncated BIO2 construct exemplified and described above with methionine synthase, and was introduced into the Arabidopsis bio2 mutant, so that the thus this form of cobalamin plays an important role in amino protein was cytosolic, it was unable to complement the mutant, acid metabolism as well as in one-carbon metabolism.29 Apart even when plants were fed with the substrate dethiobiotin.38 It from methionine synthase, B12-dependent methyltranferases play is likely that one or more mitochondrial proteins are necessary essential roles in anaerobic microbiology through participation in for biotin synthase activity, since the enzyme appears to be a methanogenesis32 and acetogenesis, where the enzymes are able large multisubunit complex. It may also reflect the need for to accept methyl groups from a broad range of donors and pass correct assembly of the Fe–S centre on the enzyme, discussed in them onto specific receptors.29 In the final class of B12-dependent more detail in the reviews by Marquet et al. and Mendel et al. reaction, B12 is associated with the reductive dehalogenation of in this issue. However, in other eukaryotic organisms, such as aromatic and aliphatic chlorinated organics. Although it appears yeast, the situation appears different. Saccharomyces cerevisiae that the reductive dehalogenation enzymes are mechanistically apparently contains only the three last enzymes of the pathway, quite distinct from either the isomerase or the methyltransferases, namely diaminopelargonic acid aminotransferase, dethiobiotin the role of cobalamin in the dehalogenation process has yet to be synthase and biotin synthase,39 whereas Schizosaccharomyces fully elucidated.29 pombe contains only the biotin synthase gene.40 As in plants,

Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. the biotin synthase activity is associated with the mitochondrial 2.2 The main differences among eukaryotic organisms compartment. Because of these truncated pathways, these two organisms are auxotrophic for biotin, and they have developed 2.2.1 Compartmentalisation. In eukaryotic cells, metabolic specific transporters allowing growth in the presence of the pathways may be split or shared between several compartments. appropriate biotin intermediates. Compartmentalisation is not necessarily the same in all eukary- c) Pantothenate. The first enzyme of pantothenate biosyn- otes. For example, plant cells have plastids, a unique compartment thesis, ketopantoate hydroxymethyltransferase (KPHMT), uses having important biosynthetic functions, which makes the distri- 5,10-methylenetetrahydrofolate as cofactor. The enzyme from bution and cellular trafficking of numerous metabolites even more Arabidopsis was found to be synthesised with an N-terminal complex in these organisms. extension with the characteristics of a mitochondrial targeting

a) Folate and C1 metabolism. Only bacteria, plants and sequence, and the terminal part of folate biosynthesis is also lower eukaryotes (yeast, protozoa) have a complete biosynthetic located in this compartment. The location of KPHMT was pathway for THF. In plants, this pathway involves the cytosol confirmed as mitochondrial using GFP fusion experiments.41 In for the synthesis of pterin, the plastids for the synthesis of p- the same paper, pantothenate synthetase, the final enzyme of aminobenzoate and the mitochondria for the assembly of the the pathway, was shown to be cytosolic, confirming earlier work different parts of the molecule33 (see also the review by Webb and that found no evidence for targeting sequences in this protein.42 Smith in this issue). In yeast and protozoa the situation is not so These results imply that either ketopantoate or pantoate must be clear, but some of the proteins that are present in the mitochondria transported out of the mitochondrion for the final step, but to of plants appear also to be associated with mitochondria in yeast.34 date nothing is known about possible transporters. In contrast,

In all eukaryotes, folate and C1 metabolism are compartmen- in the yeast S. cerevisiae, both KPHMT and pantothenate talised between the cytosol and organelles.35 In yeast and animals, synthetase have putative mitochondrial targeting sequences at the 42 the C1 units required to sustain C1 metabolism are produced from N-terminus. serine or formate in the cytosol as well as in the mitochondria. d) Moco. In plants and humans, molybdenum cofactor and the However, the syntheses of purines and dTMP are mostly present enzymes that use it are synthesised in the cytoplasm. As some of

954 | Nat. Prod. Rep., 2007, 24, 949–962 This journal is © The Royal Society of Chemistry 2007 View Article Online

the Moco-dependent enzymes are localised in the mitochondria by the glycine decarboxylase complex (GDC) and SHMT.45 In or the peroxisomes,23 they are transported after synthesis to their these reactions, GDC catalyzes the oxidative cleavage of one final cellular compartments.43 molecule of glycine, providing the 5,10-methylenetetrahydrofolate required for the conversion of a second molecule of glycine into 2.2.2 Specific needs in some eukaryotes. Some organisms serine by the reverse activity of SHMT. These two enzymatic have particular metabolic functions (for example, autotrophic systems exist in trace amounts in the mitochondria from non organisms, such as plants, have developed several metabolisms photosynthetic organisms, where they are involved in glycine specifically related to the photosynthetic activity) and might have catabolism. In leaves of C3-plants, however, where high photores- specific needs for one cofactor or another. piratory activity requires high GDC and SHMT activities, these a) Folate and C1 metabolism. In plants there is a specific pathway enzymes represent 40% of the soluble mitochondrial proteins. This associated with photosynthetic carbon assimilation and connected is possibly why the folate concentration in these mitochondria 44 to C1 metabolism: the photorespiratory pathway. This pathway is higher than in the other cellular compartments.46 It is also is initiated at the level of ribulose 1,5-bisphosphate carboxy- possible that, in C3-plants, glycine represents a potential source of lase/oxygenase (Rubisco). This is a bifunctional chloroplastic C1 units. enzyme that catalyses both the carboxylation and oxygenation of b) Cobalamin and methionine synthase. Vitamin B12 is unusual ribulose 1,5-bisphosphate. The carboxylation reaction leads to the in that its synthesis is confined to prokaryotic organisms.47 production of two molecules of 3-phosphoglycerate, whereas the Humans require this vitamin as a cofactor for methionine oxygenation reaction leads to one molecule of 3-phosphoglycerate synthase (MetH) and methylmalonyl-CoA mutase, involved in and one molecule of 2-phosphoglycolate. This oxygenation reac- the catabolism of odd-chain fatty acids. Higher plants do not tion is the primary event of a metabolic pathway called photores- make (or require) vitamin B12 because they have the alternative, piration because it is associated with the uptake of O2 and the cobalamin-independent methionine synthase called MetE. The evolution of CO2. The wasteful nature of the process has resulted in absence of vitamin B12 in higher plants means that, unlike other the evolution, in certain plant lineages, of so-called C4-metabolism, vitamins, the major source of vitamin B12 in our diets is from which avoids the oxygenation reaction of Rubisco. In leaves of animal-derived products. Thus people who follow strict vegetarian C3-plants where photorespiration occurs, there is a recycling of regimes can easily become deficient. A particularly rich dietary two molecules of 2-phosphoglycolate into one molecule of 3- source of this vitamin is seaweed or macroalgae, such as nori phosphoglycerate involving three different organelles: the chloro- (Porphyra yezoensis), which is commonly used to wrap sushi. plast, the peroxisome and the mitochondrion. The key steps of this Recent work on the metabolism of cobalamin in algae has shown pathway take place in mitochondria (Fig. 5) where glycine is either that like all other eukaryotic organisms, algae are not able to oxidised or converted into serine by two folate-dependent reactions synthesise this vitamin de novo.48 Instead, over half of all algal that are intimately coupled. These two reactions are catalyzed species are like animals, in that they have a requirement for an external source of B12, which is needed as a cofactor for MetH.48 Algae that do not need exogenous cobalamin contain MetE, like higher plants. In some cases, such as the model green alga Chlamydomonas reinhardtii, genes for both isoforms are present,

Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. and the alga can use MetH if cobalamin is present, in which case the MetE gene is turned off.48 This is analogous to the situation in E. coli, where in the absence of a supply of cobalamin MetE accumulates to 3% of cellular protein, demonstrating that the cobalamin-dependent enzyme MetH is more efficient.17 Although the dinoflagellate Phaeodactylum tricornutum has been reported to contain methylmalonyl-CoA mutase, it does not require vitamin B12 for growth, indicating that this enzyme is unlikely to be the reason for the widespread auxotrophy. Intriguingly, the levels of free cobalamin in the environment are insufficient to support the growth of auxotrophic algae, and evidence has been obtained that bacteria can supply the vitamin directly in exchange for fixed carbon, in an apparently symbiotic interaction. This may explain the observation that there is no phylogenetic relationship between those algae that require cobalamin, indicating that the loss of the MetE gene occurred multiple times during algal evolution, which in turn Fig. 5 Schematic representation of the photorespiratory pathway empha- implies the absence of selection pressure to retain the gene. A ready sising the role of the folate dependent reactions within the mitochondria. supply of micronutrients from close association with bacteria is 1) GDC: glycine decarboxylase complex; this complex is constituted of 4 subunits, the P, H, T and L proteins. The T-protein is the folate-dependent further borne out by the observation that both thiamin and biotin protein that catalyzes the transfer of the C-2 of glycine to THF, leading auxotrophy are found within the algal kingdom, although at a 49 to the release of NH3 and the formation of 5,10-CH2-THF. 2) SHMT; lower frequency. In these cases, however, it appears that the SHMT catalyzes the conversion of a second molecule of glycine and requirement for the vitamin has arisen as a result of the loss of one

5,10-CH2-THF into THF and serine. or more genes for biosynthetic enzymes.

This journal is © The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 949–962 | 955 View Article Online

3 Nutritional aspects exceptional affinity for biotin), individuals maintained on total parental nutrition, and those receiving long-term anticonvulsant The diet must provide all the micronutrients required to alleviate therapy. nutritional disorders and to promote good health. The recom- Inherited deficiencies have also been recognised, the most mended dietary allowances (RDAs) indicate for each micronu- frequent ones being a deficiency in biotinidase (the occurrence trient the minimal intake needed to avoid nutritional disorders, of which is 1 in 60 000) and holocarboxylase synthetase (HCS).56 although higher intakes might be required for optimal health. These two proteins are involved in the recycling of biotin (see Conversely, for some vitamins, excess intake can be deleterious, Fig. 6). Although these inherited deficiencies are rather rare, they as is known for vitamin A and pyridoxal (vitamin B6). Require- can have severe, even fatal, consequences if not treated. They are ments fluctuate widely depending on the micronutrient, with the mainly associated with a modification of the biotinidase and HCS recommended daily intakes ranging from a few lg (cobalamin) activities, either because the K M for biotin is increased or because to several mg (pantothenate). Deficiencies among the population the availability of free biotin is decreased. are not only found in developing countries but also in developed countries where bad food habits lead to suboptimal intakes. Indeed, micronutrient levels vary widely depending on the food source, and good diets are made of multiple sources. In the following sections the main dietary sources and the effects of a deficiency on human health are described.

3.1 Effects of deficiency on human health

a) Folate. Folate deficiency is one of the most prevalent vitamin deficiencies worldwide and leads to a number of serious diseases. Deficiency of folate would be expected to produce a reduction of the cell capacity to synthesise DNA and thus to maintain a normal rate of cell division. Indeed, a poor folate status is often correlated with a high cellular dUMP/dTMP ratio due to limiting supply of 5,10-methylenetetrahydrofolate and a decrease of dTMP synthesis. Fig. 6 The biotin cycle. Biocytin is formed from the degradation of The increased dUMP/dTMP ratio results in higher incorporation biotinylated proteins, such as holocarboxylases, histones etc. Biotin is of dUTP in DNA, which generates point mutations, single- released from biocytin by biotinidase. Depending on the pH, the bio- tinidase activity results either in its own biotinylation or in the production and double-strand DNA breaks, and ultimately chromosomal of free biotin. Either free biotin or biotin from biotinylated biotinidase 50 breakage. These damages are risk factors for a number of cancers are later used to biotinylate new proteins, through reactions catalysed by such as colorectal, breast, pancreatic, bronchial, and cervical holocarboxylase synthetases. cancer, as well as leukaemia. Cells undergoing rapid division, such as those of the bone marrow, are likely to be more affected. From The main consequence of a biotin deficiency is a multicar-

Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. this point of view, one of the most obvious consequences of folate boxylase deficiency, with expected metabolic consequences such deficiency is megaloblastic anemia, which probably results from as organic aciduria. Many other signs, such as dermatitis, con- apoptosis of erythroblasts.51 It has also been shown that some junctivitis, ataxia, different neurological disorders, developmental neural tube defects, such as spina bifida, occurring in the early delay, etc., are often associated with biotin deficiency, although period of embryogenesis, are caused by a shortage of folate.52 it is difficult to establish a direct correlation. These signs remain Inefficient synthesis of methionine may also have several mysterious at the biochemical level, and it is likely that proteins repercussions. The first one is the accumulation of homocysteine, other than carboxylases are affected. From this point of view, it has the precursor of methionine. It is generally accepted that high been shown recently that biotin controls the expression of various plasma levels of homocysteine are a primary cause of higher proteins.28 The physiological and biochemical consequences of risk for coronary and cardiovascular diseases.53 Likewise, high biotin deficiency are thus far from being completely understood. levels of homocysteine in plasma are a risk factor for dementia c) Cobalamin. Cobalamin deficiency is associated with per- and Alzheimer’s disease.54 Secondly, a low level of methionine nicious anaemia, which is a form of anaemia characterised by results in insufficient amounts of AdoMet available for all defective production of erythrocytes and the presence of mega- the methyltransferase-catalyzed reactions. Although the resulting loblasts in the bone marrow.The condition is sometimes associated problems are less well defined, they might include neuropathy due with neurological disorders. A lack of B12 is also thought to be to impaired myelin biosynthesis. Also, hypomethylation of DNA, an independent risk factor associated with neural tube defects especially hypomethylation of gene promoter regions, may alter in unborn babies, a commonality shared with folate deficiency. gene expression resulting, for example, in elevated expression of The link between folate and B12 deficiency is likely explained some oncogenes.50 by their shared responsibilities in methionine metabolism. A lack b) Biotin. The biotin requirement of most organisms is low of B12 affects the two human enzymes that require it, namely and no severe biotin deficiency has been observed in humans, methionine synthase and methylmalonyl-CoA mutase, and gives except in cases of genetic diseases.55 The well-known ‘nutritional’ rise to elevated levels of homocysteine and methylmalonic acid, biotin deficiency concerns people eating a diet very rich in raw egg- respectively. The high levels of the latter allow B12 deficiency to white (which contains a large amount of avidin, a protein with an be differentiated from simply a lack of folate.

956 | Nat. Prod. Rep., 2007, 24, 949–962 This journal is © The Royal Society of Chemistry 2007 View Article Online

The major reason for B12 deficiency is associated with problems by the mutation and should allow the synthesis of Moco. pPMP with the B12 absorption system. B12 is absorbed from the intestine is more stable than Moco itself and has an identical structure in by binding of the cobalamin to a secreted glycoprotein called all organisms. Thus, pPMP was overproduced in the bacterium E. intrinsic factor. The bound complex interacts with a specific coli and purified. MOCS1 knockout-mice with a block in the first receptor on the mucosa of the ileum, where it is internalised. B12 is step of Moco biosynthesis were created bearing a genetic defect released and transferred to another protein called transcobalamin, identical to the human patients.66 Similar to humans, heterozygous which is responsible for transport of the vitamin around the mice displayed no symptoms, but homozygous Moco-deficient body. Some congenital forms of pernicious anaemia are due to animals displayed symptoms resembling those of the human defects in this uptake system, most notably with intrinsic factor.57 deficiency state and died within ten days after birth. Due to the More significantly, some people, especially the elderly, no longer mutation, no molybdopterin or active Moco was detectable, and produce intrinsic factor, due to atrophy of the mucosa of the consequently all Mo-enzyme activities were absent. stomach, which appears to be brought about by autoimmune Repeated injections of pPMP into MOCS1-deficient mice re- factors.58 However, the deficiency in B12 can be easily treated sulted in a dose-dependent extension of life span.67 Molybdopterin by intramuscular injections of vitamin B12 or by very large oral (MPT) levels and Mo-enzyme activities were partially restored. doses of cobalamin. Stopping pPMP treatment at any time resulted in a progressive d) Pantothenate. The word pantothenate is derived from the reduction of MPT levels and Mo-enzyme activities, and death of Greek word pantos, meaning everywhere, due to the presence of the animal 10–15 days after receiving the last injection. Injection the vitamin in most foodstuffs,59 a fact that is supported by a of pPMP into these mice every second day normalised their lack of pantothenate deficiencies reported in nature. However, symptoms, and they reached adolescence and were fertile.67 It experimental induction of dietary pantothenate deficiencies, by remains to be seen whether delayed onset of the described therapy treatment with x-methylpantothenate, a metabolic antagonist, has will still allow reversal of neurological damage. As a next step, been shown to produce varying symptoms in different organisms. scaling up of pPMP production is in progress in order to have For example, animals exhibit depression, sleep disturbances, per- sufficient amounts available for clinical trials. sonality changes, cardiac instabilities, and neurological disorders such as ‘burning feet’ syndrome, amongst others. Pantothenate 3.2 Main dietary sources deficiency observed in chicks was shown to cause an outbreak of dermatitis around the eyes and beak, and resulted in the a) Folate. The recommended dietary allowance for folates is vitamin being referred to as chick anti-dermatitis factor because currently 400 lg for an adult and increases to 600 lg for pregnant treatment with pantothenate alleviated the problem.60 Due to the women (Food and Nutrition Information Center, 2004). Although absence of pantothenate deficiencies in the general population, folate is quite abundant in liver, which plays an important role in there is no minimum dietary requirement as an adequate amount folate metabolism and storage in animals, plant food is by far of pantothenate can be obtained by eating a balanced diet the biggest contributor to the folate intake of adults (Table 1). (www.foodstandards.gov.uk/multimedia/pdfs/panto.pdf). However, levels vary considerably depending on the plant species e) Moco. A mutation in the biosynthetic pathway of Moco and the nature of the tissues. has dramatic consequences for the cell because pleiotropically Folate synthesis in plants is tightly controlled and fluctuates de-

Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. all enzymes needing Mo lose their activity at the same time. In pending on the metabolic requirements. This implies that the folate humans, a combined deficiency of Mo-enzymes was first described content will vary from one tissue to another and as a function of by Duran et al.61 Babies born with this defect show feeding plant development. Generally, folate is more abundant in actively difficulties, severe and progressive neurological abnormalities, and dividing tissues, such as embryos and meristematic tissues. These dysmorphic features of the brain and head. So far, disease-causing observations fit well with a high activity of nucleotide synthesis mutations have been identified in three of the four known Moco- in rapidly dividing tissues and the utilisation of 5,10-methylene- biosynthetic genes in humans: mocs1, mocs2 and gephyrin.62 The and 10-formyltetrahydrofolate for the synthesis of thymidylate and clinical symptoms may result from the deficiency of sulfite oxidase purines. This is probably why the folate content in the embryo part that protects the organism, in particular the brain, from elevated of the seed increases further during germination and is often much levels of toxic sulfite.63 higher than in cotyledons.46 Folate concentration also increases in No therapy is currently available to cure the symptoms of this leaves during development, and this accumulation was correlated disease. Moco deficiency cannot be treated by supplementation with the build-up of the photosynthetic apparatus. The relation- with the cofactor. Moco is extremely unstable outside the pro- ship between folate accumulation in leaves and photosynthesis is tecting environment of an apo-Mo-enzyme; its half-life is only not yet understood. Part of the folate synthesised in leaves might a few minutes in aqueous solution at neutral pH.64 In addition, contribute to the photorespiratory process in mitochondria, but no chemical synthesis of Moco or any of its intermediates has most of it accumulates in a ‘cytosolic fraction’.46 In the cytosol, been successful so far, which hampers its large-scale production methyl-THF,the predominant derivative, is likely required for Met

for therapeutic use. However, very recently, a model has been synthesis and turnover of AdoMet, thus suggesting a high C1 developed that could lead to a cure of Moco-deficiency. Genetic metabolic activity associated with photosynthesis. In any case, the

analyses of patients showed that most of them had defects in relationship between photosynthesis and C1 metabolism probably the first step of Moco biosynthesis, i.e. the conversion of GTP explains why green leafy vegetables are a good source of folate. to cyclic pyranopterin monophosphate, pPMP.65 The idea was to By contrast, roots (such as carrots), storage organs (potatoes) and treat patients of this class with the missing intermediate pPMP most fruits are poor sources (Table 1). Finally, it must be kept in because the steps subsequent to pPMP formation are not affected mind that the folate content will depend on the way the food is

This journal is © The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 949–962 | 957 View Article Online

Table 1 Amounts of folate, biotin, pantothenate and cobalamin found in a range of food. Values are given in lg per 100 g food portion. Data for folate, pantothenate and cobalamin were adapted from the United States of Agriculture National Nutrient Database for Standard Reference (www.nal.usda.gov/fnic/foodcomp/search). Data for biotin were adapted from Staggs et al.107

Food Folate Biotin Pantothenate Cobalamin

Spinach, raw 193 0.7700 Broccoli, raw 62 0.95 570 0 Carrots, raw 19 0.6(canned) 273 0 Potatoes, baked 9 0.2 519 0 Orange 30 0.05 250 0 Tomatoes 15 0.7880 Lentils, cooked 181 — 630 0 Rice, white, cooked 97 — 390 0 Cow’s milk 5 0.1 362 0.44 Beef, cooked 7 4.5(hamburger) 676 1.76 Haddock, cooked 12.90.7 150 1.39 Liver (beef), cooked 260 41.6 6940 83 Egg, whole, hard-boiled 44 21.4 1400 1.10

stored, processed or cooked, the different forms of folate differing e) Moco. Molybdenum in the form of molybdate is abundant in their susceptibility to loss (for a review see ref. 68). in water and food, and hence there are no nutritional shortages b) Biotin. The recommended daily allowance of biotin is 150 lg considered. The recommended dietary allowance for molybdenum (EU) and 300 lg (US). Biotin deficiency is rare because these is currently 50 lg for an adult, and in most countries the amount relatively low requirements are generally covered by a common of molybdenum taken up by adults widely exceeds this allowance. diet. Biotin present in food (Table 1) is covalently linked to proteins. During the digestive process, these proteins are degraded 3.3 Strategies for enhancement by peptidase, and the final steps of these proteolyses lead to biocytin, i.e. biotinyl-lysine or biotinyl-lysyl peptides. Biotin is a) Folate. The fact that folate levels vary greatly in different plant liberated from these products by biotinidase, a specific enzyme species (Table 1), implies that there is a natural potential for folate present in the pancreatic juice. Biotin is then transported in enhancement. Different strategies can be followed to achieve the blood, either as free biotin (20%) or linked to the biotinidase goal of folate biofortification. These strategies have already been (80%). Once in the cell, biotin is activated as biotinyl-AMP before discussed in several reviews.33,70 In summary, they can be divided being attached to apocarboxylases (or other proteins such as into two main groups: exploiting the natural variation in folate histones), a reaction catalyzed by holocarboxylase synthetase. This levels and metabolic engineering. set of reactions (degradation of the holoenzyme by peptidases, The first approach relies on a selection process based on biotinidase activity and attachment to new carboxylases, see molecular mapping techniques: the quantitative trait loci (QTL) Fig. 6) also exists within cells. Such a cellular biotin cycle explains responsible for folate accumulation can be identified and used in

Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. the low requirement for biotin from foodstuffs, especially taking molecular-marker-assisted breeding programs. However, it must into account that some biotin is also produced by the intestinal be kept in mind that natural variations existing between different flora. varieties of one particular plant species are unlikely to be very c) Cobalamin. B12 is required in very small amounts by humans. large. Nevertheless, for plants that intrinsically have a low folate The RDA is around 2 lg per day. Dairy products are good sources level, such as rice, an increment factor of ten, at least, should of B12, as are meat and eggs.69 Vegetables do not contain B12, be reached to obtain a truly improved crop. A second difficulty as higher plants neither make nor require it for their metabolism. is that such an approach requires a simple, fast, reliable and Consequently, those who adhere to strict vegetarian diets are prone preferably high-throughput procedure for folate determination. to becoming B12-deficient, and dietary deficiency of B12 due to Several methods of folate analysis in plants have been established vegetarianism is increasing. One way to counteract this is for these (reviewed in ref. 71) but, up till now, none of them meet these people to eat certain types of macroalgae (seaweed) that are rich in requirements. B12 (see above). Prokaryotes are the world’s suppliers of vitamin The metabolic engineering approach implies genetic modifica- B12, and many enteric bacteria are able to produce cobalamin. tions of plant folate metabolism. These modifications could target Although ruminants are able to absorb the B12 produced by their the folate biosynthetic pathway itself, the reactions affecting the enteric bacteria, the human B12 uptake system does not allow the stability and storage, and the reactions involved in the breakdown uptake of the B12 produced in the lower intestine. Thus the vast and recycling of the cofactor. majority of B12 is obtained by humans from their diet. Concerning the biosynthetic pathway, a promising route is d) Pantothenate. Pantothenate is found ubiquitously in food the simultaneous enhancement of the synthesis of the two main

including meat, vegetables, mushrooms, fish and eggs, and there- folate precursors, the H2pterin and the p-aminobenzoic acid. fore there is no known dietary deficiency. Moreover, there is no This hypothesis has recently been confirmed in two independent RDA for pantothenate, and it is generally considered to be safe attempts to engineer pterin biosynthesis. These two attempts to consume in large quantities. Indeed this has led to various aimed to express GTP cyclohydrolase I (GTPCHI), the first therapeutic treatments with large doses of the vitamin, including enzyme involved in pterin synthesis. In the first experiment, the for acne and in facilitating weight loss. authors expressed in Arabidopsis the GTPCHI from E. coli,an

958 | Nat. Prod. Rep., 2007, 24, 949–962 This journal is © The Royal Society of Chemistry 2007 View Article Online

enzyme that is not subject to metabolic regulation,72 whereas in isolated. These strains, which produce pantothenate upon supple- the second experiment the authors expressed in the tomato the mentation with b-alanine, were generated by UV-induced random mammalian enzyme, which is predicted to escape feedback control mutagenesis and identification of strains resistant to several in plants.73 In both experiments the transgenic lines contained potential antimetabolites including salicylate, a-ketoisovalerate several hundred times more pterin but only two to four times and b-hydroxyaspartate.83 Overexpression of the first enzyme more folate, indicating that GTPCHI was indeed limiting but that in branched chain amino acid biosynthesis, acetohydroxyacid other factors are also regulating folate synthesis. Evidence that synthase isozyme II, led to a further increase in pantothenate synthesis of p-aminobenzoic acid is another limiting step for folate production.84 accumulation was shown by the latter group: in their attempt The most comprehensive study of the pantothenate pathway to engineer the pterin branch of folate synthesis, they observed in a single organism for the purpose of pathway engineering has that the p-aminobenzoic level in fruits was depleted following been in the commercially important Corynebacterium glutamicum. GTPCHI expression and pterin accumulation. In fact, when both Early work with this organism demonstrated that the supply of aminodeoxychorismate synthase, the first step in p-aminobenzoic b-alanine via aspartate a-decarboxylase was the limiting factor synthesis, and GTPCHI were co-expressed in tomato fruits, the in pantothenate accumulation.85 Overexpression of the native C. transgenic fruits contained 20 times more folate than the control.74 glutamicum protein leads to pantothenate production equivalent Folate derivatives are rather unstable molecules that undergo to that observed when supplying the product of the enzyme, spontaneous oxidative degradation. However, the stability of the b-alanine. Sahm and Eggeling enhanced the flux through the cofactor is considerably increased when bound to folate-dependent other branch of the biosynthetic pathway by overexpressing proteins. Thus, stabilisation of folate can possibly be achieved by genes for valine biosynthesis (ilvBNCD)intandemwiththe over-expressing folate-binding proteins (FBP) in plant cells, such C. glutamicum panBC operon in a strain unable to synthesise as the mammalian FBP found in milk.75 Other proteins having isoleucine (ilvA).86 In the presence of exogenous b-alanine this a high affinity for folate, for instance the T-protein of GDC,76 strainwasabletoaccumulateupto1gl−1 of pantothenate. The could also be tested. These strategies would not only stabilise third step in the biosynthetic pathway (ketopantoate reductase) folate but also create a folate sink, resulting presumably in a is solely encoded by ilvC in this organism, and so the tandem

stimulation of synthesis. Re-routing of C1 metabolism towards overexpression of the valine biosynthetic pathway also enhanced accumulation of the most stable folate derivatives may be yet the rate of this transformation.87 A metabolic network analysis another approach to stabilise the folate pool. The most stable of this overproduction strain demonstrated that the flux from the

form of folate is 5-formyltetrahydrofolate. No function in C1 branch point between valine and pantothenate biosynthesis was metabolism has been assigned to this molecule so far: it is thought 10-fold more favourable for valine biosynthesis. Chassagnole and to play a role as a storage form or to have a regulatory function, colleagues attempted to increase the proportion of pantothenate

inhibiting some enzymes of C1 metabolism, such as mitochondrial over valine by the use of nitrogen-limiting conditions, but this led SHMT. The only enzyme that uses 5-formyltetrahydrofolate is 5- to the production of a range of non-nitrogenous compounds and formyltetrahydrofolate cycloligase (5-FCL), which catalyzes the accumulation of glycine.88,89 This suggests that the limiting factor ATP-dependent reverse conversion of 5-formyltetrahydrofolate to might be the regeneration of 5,10-methyleneTHF for ketopantoate 5,10-methenyltetrahydrofolate.77 Recently, an Arabidopsis 5-FCL synthesis. More recently, the addition of an ilvE mutation, to 78 Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. knockout mutant has been characterised, but only a two-fold prevent valine biosynthesis, together with multiple copies of the increase was observed in the folate pool. panBC operononanexpressionvector,havebeenusedtoincrease Reducing the rate of folate catabolism might also lead to higher the pantothenate levels further.90 As yet, however, the levels folate accumulation. Indeed, it was recently shown that plants achieved do not match those observed in the E. coli overproduction can have high folate-breakdown rates, approximately 10% per day. strains. This breakdown involves oxidative cleavage of the molecule, giving c) Cobalamin. Cobalamin is one of the few vitamins that p-aminobenzoylglutamate and pterin. However, most of these is produced commercially by bacterial fermentation.91 This is degradation products are recycled as folate precursors,79 and it because the chemical synthesis of the vitamin is far too com- is not certain that engineering the catabolic pathway alone would plex to contemplate for industry. There has thus been some be sufficient to significantly increase the folate concentration. significant research into investigating the molecular genetics and b) Pantothenate. The overexpression of elements of the pan- biochemistry of cobalamin biosynthesis with a view towards tothenate pathway as a mechanism for increased vitamin produc- enhancing the production of B12 by bacteria. Indeed, it was this tion has been explored in several organisms. Enhanced expression approach developed by the Rhone-Poulencˆ company that led to of pantothenate to some extent was achieved by the overexpression the elucidation of the aerobic biosynthetic pathway for vitamin of three of the four enzymes: in E. coli K12 ketopantoate reductase B12,92 and which allowed the company to take out several patents overexpression leads to increases in cellular pantothenate levels,80 on maximising cobalamin synthesis. whilst in Salmonella enterica serovar Typhimurium overexpression The research undertaken by Rhone-Poulencˆ gave rise to a of ketopantoate hydroxymethyltransferase is sufficient to increase production strain of Pseudomonas denitrificans, which was gen- cellular pantothenate levels.81 In plants, the supply of b-alanine, erated using the acquired information on cobalamin biosynthesis. which in these organisms is via a different pathway, seems to Over a two year period, this resulted in an increase in B12 in be a limiting factor. Overexpression of the bacterial aspartate P. denitrificans of approximately 100-fold. There are over thirty a-decarboxylase in tobacco leaves resulted in an increase in genes involved in cobalamin biosynthesis and the detailed patents both b-alanine and pantothenate levels.82 Strains of E. coli that suggest that at least 10 of these genes have had their copy accumulate up to 65 g l−1 of pantothenate in culture have been number increased. For instance, it was noted that increasing the

This journal is © The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 949–962 | 959 View Article Online

cobF–cobM gene cluster increased cobalamin production by 30%, or vitamin PP or vitamin B3) are the precursors of NAD(P) whereas amplification of cobA and cobE resulted in a further 20% synthesis in animals. They are also involved, in plants, in the enhancement. Moreover, it is likely that gene transcription and synthesis of pyridine alkaloids (ricinine, nicotine, trigonelline). translation elements were modified to enhance the production of The pathways of NAD(P) synthesis and recycling in plants are the pathway enzymes. The researchers also looked to engineer the starting to emerge and implicate the plastids, the cytosol and pathway by replacing one of the key regulatory enzymes of the possibly the mitochondria.102 Thiamine pyrophosphate (vitamin pathway that displayed substrate inhibition with a variant that no B1) synthesis is poorly known in the plant kingdom. What is longer had this unwanted characteristic. Thus enhanced flux along known is that thiamine pyrophosphate is synthesised through two the pathway could be achieved by replacing the uroporphyrinogen different branches, one for the synthesis of the pyrimidine moiety methyltransferase with a variant from a methanogen. One of the and another for the synthesis of the thiazole moiety. It is not limiting factors for cobalamin production is the synthesis of the clear whether the thiazole moiety is synthesised in chloroplasts or lower axial ligand, dimethylbenzimidazole (DMB). The Rhone-ˆ mitochondria,103 but it has been clearly shown that the enzyme Poulenc researchers found that overproducing a protein called catalyzing the condensation of thiazole phosphate and pyrimidine BluB gave rise to significantly enhanced DMB production and pyrophosphate to produce thiamine monophosphate is exclusively increased the yield of B12.91 Only recently has it been shown located in the chloroplasts.104 that BluB is able to catalyse a remarkable oxygen-dependent Taking these new data into account, it is obvious that transport transformation of reduced flavin into DMB.93 This combined of coenzymes from one compartment to another might play approach of basic science and applied biotechnology allowed an important role in the regulation of the pathways. Transport Rhone-Poulencˆ to produce a strain that accounts for about 80% systems are always difficult to study, and this is particularly true of the world’s production of cobalamin. when the carriers are present in very low amounts, as is presumably the case for those involved in the transport of coenzymes. With the exception of two folate transporters recently identified, both 4 Conclusion: compartmentalisation, a challenging located in the envelope of chloroplasts,105,106 carriers involved in area vitamin transport are not known in plants. As pointed out above, these transport systems are potential limiting steps for vitamin Over the course of this last decade, much progress has been made synthesis, distribution and storage, and will probably be important in understanding how coenzymes are synthesised. Indeed, most of keys in most of the strategies used to engineer vitamin content in the pathways involved in the biosynthesis of these molecules are plants. now established in bacteria, lower eukaryotes and plants, although the mechanisms of a fair number of the required reactions remain to be understood in detail. Identification of the genes involved in 5 References the various steps of these metabolic routes open new possibilities 1E.A.Cossins,Can. J. Bot., 2000, 78, 691–708. of manipulating (engineering) the coenzyme content of various 2 J. M. Mouillon, S. Aubert, J. Bourguignon, E. Gout, R. Douce and F. organisms, either for chemical production of these molecules in Rebeill´ e,´ Plant J., 1999, 20, 197–205. bacteria or to improve nutrition by modifying levels in plants. 3 A. U. Igamberdiev, N. V.Bykova and L. A. Kleczkowski, Plant Physiol. Biochem., 1999, 37, 503–513. Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. However, the situation in eukaryotes is more complex than in 4 S. Ravanel, R. Douce and F. Rebeill´ e,´ ‘The uniqueness of tetra- bacteria because of the subcellular compartmentalisation of the hydrofolate synthesis and one-carbon metabolism in plants’, in Plant pathways, which imposes the need to take into account the Mitochondria: From Genome To Function, ed. D. A. Day, A. H. Miller transport of coenzymes, intermediates and precursors from one and J. Whelan, Kluwer Academic, Dordrecht, 2004, pp. 277– 292. compartment to another. 5 P. M. Smith and C. A. Atkins, Plant Physiol., 2002, 128, 793–802. As a matter of fact, once synthesised in a given compartment, 6 M. Neuburger, F. Rebeill´ e,´ A. Jourdain, S. Nakamura and R. Douce, coenzymes have to be transported to the other cellular territories J. Biol. Chem., 1996, 271, 9466–9472. 7 A. D. Hanson and S. Roje, Annu. Rev. Plant Physiol. Plant Mol. Biol., where they are required. In plants, all the cellular compartments 2001, 52, 119–137. are more or less involved in vitamin biosynthesis. Some of these 8 S. Ravanel, M. A. Block, P. Rippert, S. Jabrin, G. Curien, F. Rebeill´ e´ syntheses are restricted to one compartment, but it is remarkable and R. Douce, J. Biol. Chem., 2004, 279, 22548–22557. that vitamin biosyntheses in plants are often split between 9 N. J. Krogan, M. Kim, A. Tong, A. Golshani, G. Cagney, V.Canadien, D. P. Richards, B. K. Beattie, A. Emili, C. Boone, A. Shilatifard, different compartments. So, pyridoxal phosphate (vitamin B6) S. Buratowski and J. Greenblatt, Mol. Cell. Biol., 2003, 23, 4207– is synthesised in two steps, exclusively located in the cytosolic 4218. compartment.94 Riboflavin (vitamin B2) synthesis is probably 10 R. A. Martienssen and E. J. Richards, Curr. Opin. Genet. Dev., 1995, restricted to the plastids,95 and this also holds true for tocopherols 5, 234–242. 11 R. Surtees, J. Leonard and S. Austin, Lancet, 1991, 338, 1550–1554. 96 97 (vitamin E) and carotenoids (pro-vitaminA). However, as 12 T. Tolstykh, J. Lee, S. Vafai and J. B. Stock, EMBO J., 2000, 19, mentioned above, the first step in pantothenate (vitamin B5) 5682–5691. biosynthesis is located within mitochondria whereas the next steps 13 R. G. Matthews, ‘Methionine biosynthesis’, in Folates and Pterins, ed. 98 R. L. Blakley and S. J. Benkovic, Wiley Interscience, New York, 1984, are in the cytosol. In contrast, the first steps in biotin (vitamin pp. 497–553, ,. B8) synthesis are in the cytosolic compartment, but the last one 14 M. T. Croft, A. D. Lawrence, E. Raux-Deery, M. J. Warren and A. G. is associated with mitochondria,99 and this also holds true for Smith, Nature, 2005, 438, 90–93. ascorbate (vitamin C).100 As also described here, the synthesis 15 R. G. Matthews, Acc. Chem. Res., 2001, 34, 681–689. 16 R. Pejchal and M. L. Ludwig, PLOS Biol., 2005, 3, 254–265. of folate (vitamin B9) is shared between cytosol, chloroplast and 17 J. C. Gonzalez, R. V. Banerjee, S. Huang, J. S. Sumner and R. G. mitochondria.101 Nicotinate or nicotinamide (also known as niacin Matthews, Biochemistry, 1992, 31, 6045–6056.

960 | Nat. Prod. Rep., 2007, 24, 949–962 This journal is © The Royal Society of Chemistry 2007 View Article Online

18 J. C. Evans, D. P. Huddler, M. T. Hilgers, G. Romanchuk, R. G. 60 C. M. Smith and W. O. Song, J. Nutr. Biochem., 1996, 7, 312–321. Matthews and M. L. Ludwig, Proc. Natl. Acad. Sci. U. S. A., 2004, 61 M. Duran, F. A. Beemer, C. van de Heiden, J. Korteland, P. K. de 101, 3729–3736. Bree, M. Brink, S. K. Wadman and I. Lombeck, J. Inherited Metab. 19 T. P. Begley, C. Kinsland and E. Strauss, Vitam. Horm., 2001, 61, Dis., 1978, 1, 175–178. 157–171. 62 J. Reiss and J. L. Johnson, Hum. Mutat., 2003, 21, 569–576. 20 D. E. Cane and C. T. Walsh, Chem. Biol., 1999, 6, R319–R325. 63 J. L. Johnson and M. Duran, in The Metabolic and Molecular Bases 21 H. G. Wood and R. E. Barden, Annu. Rev. Biochem., 1977, 46, 385– of Inherited Disease, ed. C. Scriver , A. L. Beaudet, W. S. Sly and 413. D. Valle, McGraw–Hill, New York, 2001, pp. 3163–3177. 22 E. I. Stiefel, Met. Ions Biol. Syst., 2002, 39, 1–29. 64 S. Kramer, R. V. Hageman and K. V. Rajagopalan, Arch. Biochem. 23 G. Schwarz and R. R. Mendel, Annu. Rev. Plant Biol., 2006, 57, 623– Biophys., 1984, 233, 821–829. 647. 65 J. Reiss, Hum. Genet., 2000, 106, 157–163. 24 R. Hille, Arch. Biochem. Biophys., 2005, 433, 107–116. 66 H. J. Lee, I. M. Adham, G. Schwarz, M. Kneussel, J. O. Sass, W.Engel 25 C. L. Thompson and A. Sancar, Oncogene, 2002, 21, 9043–9056. and J. Reiss, Hum. Mol. Genet., 2002, 11, 3309–3317. 26 C. M. Bray and C. E. West, New Phytol., 2005, 168, 511–528. 67 G. Schwarz, J. A. Santamaria-Araujo, S. Wolf, H. J. Lee, I. M. Adham, 27 J. Zempleni, Annu. Rev. Nutr., 2005, 25, 175–196. H. J. Grone, H. Schwegler, J. O. Sass, T. Otte, P. Hanzelmann, R. R. 28 K. Dakshinamurti and S. Litvak, J. Biol. Chem., 1970, 245, 5600–5605. Mendel, W. Engel and J. Reiss, Hum. Mol. Genet., 2004, 13, 1249– 29 R. Banerjee and S. W. Ragsdale, Annu. Rev. Biochem., 2003, 72, 209– 1255. 247. 68 J. Scott, F. Rebeill´ e´ and J. Fletcher, J. Sci. Food Agric., 2000, 80, 30 E. N. G. Marsh and C. L. Drennan, Curr. Opin. Chem. Biol., 2001, 5, 795–824. 499–505. 69 S. P. Stabler and R. H. Allen, Annu. Rev. Nutr., 2004, 24, 299–326. 31 J. R. Roth, J. G. Lawrence and T. A. Bobik, Annu. Rev. Microbiol., 70 G. J. C. Basset, E. P.Quinlivan, J. F. Gregory and A. D. Hanson, Crop 1996, 50, 137–181. Sci., 2005, 45, 449–453. 32 R. K. Thauer, Microbiology (Reading, U. K.), 1998, 144, 2377–2406. 71 S. Storozhenko, S. Ravanel, G.-F. Zhang, F. Rebeill´ e,´ W.Lambert and 33 F. Rebeille, S. Ravanel, S. Jabrin, R. Douce, S. Storozhenko and D. D. Van Der Straeten, Trends Food Sci. Technol., 2005, 16, 271–281. Van Der Straeten, Physiol. Plant., 2006, 126, 330–342. 72 T. Hossain, I. Rosenberg, J. Selhub, G. Kishore, R. Beachy and K. 34 U. Guldener, G. J. Koehler, C. Haussmann, A. Bacher, J. Kricke, D. Schubert, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 5158–5163. Becher and J. H. Hegemann, Mol. Biol. Cell, 2004, 15, 3811–3828. 73 R. Diaz de la Garza, E. P. Quinlivan, S. M. Klaus, G. J. Basset, J. F. 35 K. E. Christensen and R. E. MacKenzie, Bioessays, 2006, 28, 595–605. Gregory, 3rd and A. D. Hanson, Proc. Natl. Acad. Sci. U. S. A., 2004, 36 R. Boldt and R. Zrenner, Physiol. Plant, 2003, 117, 297–304. 101, 13720–13725. 37 V. Pinon, S. Ravanel, R. Douce and C. Alban, Plant Physiol., 2005, 74 R. Diaz De La Garza, J. Gregory III and A. D. Hanson, Proc. Natl. 139, 1666–1676. Acad. Sci. U. S. A., 2007, 104, 4218–4222. 38 N. Arnal, C. Alban, M. Quadrado, O. Grandjean and H. Mireau, 75 M. L. Jones and P. F. Nixon, J. Nutr., 2002, 132, 2690–2694. Plant Mol. Biol., 2006, 62, 471–479. 76 F. Rebeille, M. Neuburger and R. Douce, Biochem. J., 1994, 302(1), 39 V. Phalip, I. Kuhn, Y. Lemoine and J. M. Jeltsch, Gene, 1999, 232, 223–228. 43–51. 77 S. Roje, M. T. Janave, M. J. Ziemak and A. D. Hanson, J. Biol. Chem., 40 V. Phalip, Y. Lemoine and J. M. Jeltsch, Curr. Microbiol., 1999, 39, 2002, 277, 42748–42754. 348–0350. 78 A. Goyer, E. Collakova, R. Diaz de la Garza, E. P. Quinlivan, J. 41 H. H. Ottenhof, J. L. Ashurst, H. M. Whitney, S. A. Saldanha, F. Williamson, J. F. Gregory, 3rd, Y. Shachar-Hill and A. D. Hanson, Schmitzberger, H. S. Gweon, T. L. Blundell, C. Abell and A. G. Smith, J. Biol. Chem., 2005, 280, 26137–26142. Plant J., 2004, 37, 61–72. 79G.Orsomando,G.G.Bozzo,R.D.delaGarza,G.J.Basset,E.P. 42 U. Genschel, C. A. Powell, C. Abell and A. G. Smith, Biochem. J., Quinlivan, V. Naponelli, F. Rebeille, S. Ravanel, J. F. Gregory and 1999, 341, 669–678. A. D. Hanson, Plant J., 2006, 46, 426–435. 43 K. Nowak, N. Luniak, C. Witt, Y. Wustefeld, A. Wachter, R. R. 80 F. Elischewski, A. Puhler and J. Kalinowski, J. Biotechnol., 1999, 75, Mendel and R. Hansch, Plant Cell Physiol., 2004, 45, 1889–1894. 135–146. 44 J. Bourguignon, F. Rebeille and R. Douce, ‘Serine and glycine 81 A. Rubio and D. M. Downs, J. Bacteriol., 2002, 184, 2827–2832. Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55. metabolism in higher plants’, in Plant Amino Acids, ed. B. K. Singh, 82 W. M. Fouad and B. Rathinasabapathi, Plant Mol. Biol., 2006, 60, Marcel Dekker, New York, 1999, pp. 111–146. 495–505. 45 R. Douce, J. Bourguignon, M. Neuburger and F. Rebeill´ e,´ Trends 83 Y. Hikichi, T. Moriya, I. Nogami, H. Miki and T. Yamaguchi, Patent Plant Sci., 2001, 6, 167–176. No. EP-A 0 590 857, 1994. 46 S. Jabrin, S. Ravanel, B. Gambonnet, R. Douce and F. Rebeill´ e,´ Plant 84 T. Moriya, Y. Hikichi, Y. Moriya, T. Yamaguchi, Patent No. WO Physiol., 2003, 131, 1431–1439. 97/10340, 1997. 47 M. J. Warren, E. Raux, H. L. Schubert and J. C. Escalante-Semerena, 85 N. Dusch, A. Puhler and J. Kalinowski, Appl. Environ. Microbiol., Nat. Prod. Rep., 2002, 19, 390–412. 1999, 65, 1530–1539. 48 M. T. Croft, A. D. Lawrence, E. Raux-Deery, M. J. Warren and A. G. 86 H. Sahm and L. Eggeling, Appl. Environ. Microbiol., 1999, 65, 1973– Smith, Nature, 2005, 438, 90–93. 1979. 49 M. T. Croft, M. J. Warren and A. G. Smith, Eukaryotic Cell, 2006, 5, 87 M. Merkamm, C. Chassagnole, N. D. Lindley and A. Guyonvarch, 1175–1183. J. Biotechnol., 2003, 104, 253–260. 50 Y. I. Kim, J. Nutr. Biochem., 1999, 10, 66–88. 88 C. Chassagnole, F. Letisse, A. Diano and N. D. Lindley, Mol. Biol. 51 G. M. Li, S. R. Presnell and L. Gu, J. Nutr. Biochem., 2003, 14, 568– Rep., 2002, 29, 129–134. 575. 89 C. Chassagnole, A. Diano, F.Letisse and N. D. Lindley, J. Biotechnol., 52 J. Geisel, J. Perinat. Neonat. Nurs., 2003, 17, 268–279. 2003, 104, 261–272. 53 P. J. Stover, Nutr. Rev., 2004, 62, S3–S12. 90 A. T. Huser, C. Chassagnole, N. D. Lindle, M. Merkamm, A. 54 S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques, I. H. Rosenberg, R. B. Guyonvarch, V. Elisakova, M. Patek, J. Kalinowski, I. Brune, A. D’Agostino,P.W.WilsonandP.A.Wolf,N. Engl. J. Med., 2002, 346, Puhler and A. Tauch, Appl. Environ. Microbiol., 2005, 71, 3255–3268. 476–483. 91 J. H. Martens, H. Barg, M. J. Warren and D. Jahn, Appl. Microbiol. 55 A. Marquet, B. T. Bui and D. Florentin, Vitam. Horm., 2001, 61, Biotechnol., 2002, 58, 275–285. 51–101. 92 F. Blanche, B. Cameron, J. Crouzet, L. Debussche, D. Thibaut, M. 56 B. Wolf, J. Nutr. Biochem., 2005, 16, 441–445. Vuilhorgne, F. J. Leeper and A. R. Battersby, Angew. Chem., Int. Ed. 57 W.A. Fenton and L. E. Rosenberg, ‘Inherited disorders of cobalamin Engl., 1995, 34, 383–411. transport and metabolism’, in The Metabolic and Molecular Bases 93 M. E. Taga, N. A. Larsen, A. R. Howard-Jones, C. T. Walsh and G. C. of Inherited Disease, ed. C. R. Scriver, A. L. Beaudet, W. S. Sly and Walker, Nature, 2007, 446, 449–453. D. Valle, McGraw–Hill, New York, 1995, pp. 3129–3149. 94 M. Tambasco-Studart, O. Titiz, T. Raschle, G. Forster, N. Amrhein 58 I. Chanarin, Br. J. Haematol., 2000, 111, 407–415. andT.B.Fitzpatrick,Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 59 J. B. Tarr, T. Tamura and E. L. R. Stokstad, Am. J. Clin. Nutr., 1981, 13687–13692. 34, 1328–1337. 95 F. J. Sandoval and S. Roje, J. Biol. Chem., 2005, 280, 38337–38345.

This journal is © The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 949–962 | 961 View Article Online

96 P. Rippert, C. Scimemi, M. Dubald and M. Matringe, Plant Physiol., 102 G. Noctor, G. Queval and B. Gakiere, J. Exp. Bot., 2006, 57, 1603– 2004, 134, 92–100. 1620. 97 H. van den Berg, R. Faulks, H. F. Granado, J. Hirschberg, B. 103 S. M. Chabregas, D. D. Luche, M. A. Van Sluys, C. F. M. Menck and Olmedilla, G. Sandmann, S. Southon and W.Stahl, J. Sci. Food Agric., M. C. Silva, J. Cell Sci., 2003, 116, 285–291. 2000, 80, 880–912. 104 I. Ajjawi, Y. Tsegaye and D. Shintani, Arch. Biochem. Biophys., 2007, 98 K. M. Coxon, E. Chakauya, H. H. Ottenhof, H. M. Whitney, T. L. 459, 107–114. Blundell, C. Abell and A. G. Smith, Biochem. Soc. Trans., 2005, 33, 105 M. Bedhomme, M. Hoffmann, E. A. McCarthy, B. Gambonnet, R. G. 743–746. Moran, F. Rebeille and S. Ravanel, J. Biol. Chem., 2005. 99 A. Picciocchi, R. Douce and C. Alban, J. Biol. Chem., 2003, 278, 106 S. M. J. Klaus, E. R. S. Kunji, G. G. Bozzo, A. Noiriel, R. D. 24966–24975. de la Garza, G. J. C. Basset, S. Ravanel, F. Rebeille, J. F. 100 C. G. Bartoli, G. M. Pastori and C. H. Foyer, Plant Physiol., 2000, Gregory and A. D. Hanson, J. Biol. Chem., 2005, 280, 38457– 123, 335–343. 38463. 101 F. Rebeille, S. Ravanel, S. Jabrin, R. Douce, S. Storozhenko and 107 C. G. Staggs, W. M. Sealey, B. J. McCabe, A. M. Teague and D. M. D. Van Der Straeten, Physiol. Plant., 2006, 126, 330–342. Mock, J. Food Compos. Anal., 2004, 17, 767–776. Published on 20 August 2007. Downloaded by Harvard University 19/11/2013 20:46:55.

962 | Nat. Prod. Rep., 2007, 24, 949–962 This journal is © The Royal Society of Chemistry 2007