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Molybdenum metabolism in plants Cite this: Metallomics, 2013, 5, 1191

ńez,w Alejandro Chamizo-Ampudia, Aurora Galva ń, Manuel Tejada-Jime ´ ngel Llamas ńdez* and A Emilio Ferna The viability of plants relies on molybdenum, which after binding to the organic moiety of molybdopterin forms the molybdenum cofactor (Moco) and acquires remarkable redox properties. Moco is in the active site of critical molybdoenzymes, which use to work as small electron transport chains and participate in N

Received 20th March 2013, Accepted 6th June 2013

and S metabolism, hormone biosynthesis, toxic compound transformations and other important processes

DOI: 10.1039/c3mt00078h

here, with special attention to two main aspects, the different molybdate transporters that with a very

not only in plants but also in all the other kingdoms of life. Molybdate metabolism in plants is reviewed high affinity participate in molybdenum acquisition and the recently discovered Moco enzyme

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amidoxime-reducing component. Their functionality is starting to be understood.

Departamento de Bioquı´mica y Biologıá Molecular, Facultad de Ciencias, ´rdoba, Campus de Excelencia Internacional Agroalimentario Universidad de Co ´rdoba, Spain. (CeiA3), Campus de Rabanales, Edif. Severo Ochoa, 14071 Co E-mail: [email protected]; Fax: +34-957218591 ¨t zu Ko ¨ln, Zu ¨r Biochemie, Universita ¨lpicher Str. 47, † Present address: Institut fu ¨ln, Germany. 50674 Ko

in solution at pH higher than 4.2,1 and therefore cells take up Mo from the external medium in the form of molybdate. Mo is present in low and diverse amounts in continental (5 nmol L1) and marine (100 nmol L1) waters, and in soils (1.1 mg kg1).2–6 In biological tissues, Mo is one of the scarcest elements. In plant cells Mo is present at an average concentration of 0.2 mg kg1 dry weight,7 although it strictly depends on the availability of molybdate in soils.8 The importance of Mo for living organisms was identified in 1939 using tomato plants grown in a fully defined nutrient solution.9 Plants grown in the absence of Mo showed an impaired growth, this phenotype was avoided by addition of trace amounts of Mo. Later Mo was found as a pterin-cofactor,

Manuel Tejada-Jimeńez received his PhD degree in 2007 in Biochemistry and Molecular Biology at the University of Cordoba (Spain). His research has focused on molybdate transport as a part of molybdenum metabolism in the green alga Chlamydomonas reinhardtii. Dr Tejada-Jimeńez ¨nter joined the group of Prof. Gu Schwarz in 2010 at the Institute of Biochemistry, University of ńez Manuel Tejada-Jime Cologne (Germany), funded initially by a Marie Curie Fellowship (European Seventh Framework Programme). He is currently working on the molybdate metabolism in human cells.

Alejandro Chamizo-Ampudia finished his university studies in Biochemistry in 2009. He is carrying out the PhD studies in Biochemistry and Molecular Biology at the University of Cordoba (Spain). His research focuses on the molybdoenzymes, especially the ARC protein in the green alga Chlamydomonas reinhardtii, in the group of ńdez. Alejandro Emilio Ferna Chamizo-Ampudia has stayed Alejandro Chamizo-Ampudia for three months in the group of ¨nter Schwarz at the Institute of Biochemistry, University of Prof. Gu Cologne (Germany). In 2010, he got an FPU scholarship, one of the most prestigious PhD scholarships available in Spain.

Bioavailability and biological importance of molybdenum Molybdenum (Mo) is the only second-row transition metal with biological activity. Among the existing compounds of Mo in nature, oxyanion molybdate (MoO42) is the predominant form

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The Royal Society of Chemistry 2013

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Tutorial Review in the active centre of more than fifty enzymes involved in key oxidation–reduction reactions of nitrogen and sulphur metabolism, phytohormone biosynthesis and detoxification of xenobiotics. This catalytic activity converts Mo into an essential micronutrient for almost all living beings. Most of these Mo-containing enzymes occur in prokaryotes while only five of them have been identified in plants: nitrate reductase (NR), sulphite oxidase (SO), aldehyde oxidase (AO), xanthine dehydrogenase (XDH) and amidoxime reducing component (ARC).10 Because of the low but sustained abundance of molybdate and the efficiency of Mo-transporters, symptoms of Mo deficiency are difficult to find in plants that, if appear, derive from the reduced activity of molybdoenzymes and might affect primarily plant growth.11,12 The phenotype observed in Mo-deficient plants is characterized by an altered morphology of leaves, poor development of seeds, impairment of flower production

ń got her PhD at the Aurora Galva University of Sevilla (Spain) on the study of calcium ATPases from animal endoplasmic reticulum. At the end of the 1980’s she studied calcium regulation and structural organization in lens plasma membranes at Charles Louis’ lab (University of Minnesota, USA). Then she joined the Department of Biochemistry and Molecular Biology at the University of ń Aurora Galva Cordoba, where she is a Professor interested in nitrate sensing for nitrate assimilation and in molybdenum homeostasis using algal cells as a model system.

ńdez got his PhD Emilio Ferna from the University of Sevilla (Spain) on the characterization of nitrate reductase mutant strains in the green alga Chlamydomonas. In 1983 he worked in Rene Matagne’s lab (University of Liege, Belgium) performing genetic studies in Chlamydomonas. From 1986– 1988 he cloned the nitrate reductase gene of the alga to develop a transformation system in Pete ńdez Emilio Ferna Lefebvre’s lab (University of ńdez is Professor of Biochemistry and Minnesota, USA). Dr Ferna Molecular Biology at the University of Cordoba and his research interests are focused on sensing mechanisms of nitrate assimilation and molybdenum metabolism in Chlamydomonas.

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Metallomics and decrease in the overall plant growth.4 The effects of Mo deficiency have been studied in detail in Arabidopsis thaliana using a molybdate transport mutant.13 Transcription profiles of almost four hundred genes were found to be changed under Mo-deficient conditions, including induction of NR, the transcription factor MYB or the sulphate transporter SULTR3;5; and the inhibition of the serine carboxypeptidase SCPL13. Metabolite profiles of amino acids, sugars, organic acids, purine compounds are also altered significantly in Mo-deficient plants.13 Effects of Mo deficiency have also been reported in mammals resulting in a severe neurological degeneration, which leads to premature death within weeks after birth.14

Molybdenum transport and homeostasis in plants An accurate and well-coordinated process of Mo homeostasis is crucial for plants in order to: (i) cope with variations in molybdate availability in soils caused by environmental changes and (ii) ensure a proper molybdate supply to meet the cellular needs of Mo. In the last few years different plant proteins have been related to molybdate transport (Tables 1 and 2). However Mo homeostasis in plants remains unclear since key steps of this process such as Mo export and storage are still not well characterized (Fig. 1).

Table 1

Transport systems involved in molybdate uptake in plants

Transporter Specific anion Km MOT1;1 MOT1;2 MOT2 SHST1

MoO4

Other substrates Organism

2

7–20 nM WO42

2

2

MoO4 MoO42 SO42

n.a. 550 nM 10 mM

WO4 WO42 MoO42

C. reinhardtii A. thaliana A. thaliana C. reinhardtii S. hamata

n.a.: data not available.

Angel Llamas got his PhD in 2002 in the University of Cordoba (Spain) studying the nitrate assimilation pathway with the green alga Chlamydomonas in ńdez’s Emilio Ferna lab. Afterwards, he stayed for 30 months in Braunschweig University (Germany) in Ralf Mendel’s lab to study the Arabidopsis protein CNX1 for molybdenum cofactor synthesis. Then, in 2008 he got a Ramon y ńgel Llamas A Cajal contract, one of the most prestigious post-doc positions available in Spain. Since 2012, Dr Llamas has been tenured Professor of Biochemistry and Molecular Biology at the University of Co´rdoba and his research is focused on the molybdenum cofactor biosynthesis in eukaryotes.

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Table 2

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Molybdoenzymes and molybdate transporter proteins in plants

Organism

Protein

Identification number

Location (chromosome)

Domains

Length (aa)

Partners

Chlamydomonas reinhardtii

NR SO XDH ARC MOT1;1 MOT2

XP_001696697 XP_001701253 EDP03026 AEI61922 A6YCJ2 AEY68285

c

(9) p (3) c (12) u (9) u (4) u (13)

Moco, Cyt b5, Cytb5-R Cyt b5, Moco 2Fe–S, FAD, Moco b-Barrel, Moco u u

882 605 1304 329 519 535

No Cyt-c No Cytb5, Cytb5-R u u

Arabidopsis thaliana

NR1 NR2 SO XDH1 XDH2 AO1 AO2 AO3 AO4 ARC1 ARC2 MOT1;1 MOT1;2 MOT2

NP_177899 NP_174901 AEE73732 NP_195215 NP_195216 NP_851049 NP_189946 NP_180283 NP_563711 NP_174376 NP_199285 NP_180139 BAF01113 NP_567786

c c

(1) (1) p (3) c (4) c (4) c (5) c (3) c (2) c (1) u (1) u (5) m& p (2) v (1) u (4)

Moco, Cyt b5, Cytb5-R Moco, Cyt b5, Cytb5-R Moco 2Fe–S, FAD, Moco 2Fe–S, FAD, Moco 2Fe–S, FAD, Moco 2Fe–S, FAD, Moco 2Fe–S, FAD, Moco 2Fe–S, FAD, Moco b-Barrel, Moco b-Barrel, Moco u u u

917 917 393 1361 1353 1368 1321 1332 1337 318 308 456 464 460

No No Cyt b5, Cyt-c No No No No No No u u u u u

Oryza sativa Japhonica

NR SO XDH AO ARC1 ARC2 MOT1;1 MOT1;2 MOT2

NP_001048253 ABA97730 NP_001050420 NP_001064133 NP_001063929 NP_001063930 BAD03554 NP_001043703 NP_001048746

c

(2) p (12) c (3) c (10) u (9) u (9) u (8) u (1) u (3)

2Fe–S, FAD, Moco Moco 2Fe–S, FAD, Moco 2Fe–S, FAD, Moco b-Barrel, Moco b-Barrel, Moco u u u

889 400 1369 1387 324 319 455 463 457

No Cyt b5, Cyt-c No No u u u u u

Populus trichocarpa

NR SO XDH AO1 AO2 ARC MOT1;1 MOT2

XP_002307415 XP_002300104 XP_002314067 XP_002313633 XP_002328085 ABK95960 XP_002308631 XP_002318449

c

2Fe–S, FAD, Moco Moco 2Fe–S, FAD, Moco 2Fe–S, FAD, Moco 2Fe–S, FAD, Moco b-Barrel, Moco u u

899 393 1368 1372 1371 325 450 459

No Cyt b5, Cyt-c No No No u u u

(LGV) p (LGI) c (LGIX) c (LGIX) c (u) u (LGIII) u (—) u (—)

For this table the NCBI (http://www.ncbi.nlm.nih.gov/protein) and WolF PSORT (http://wolfpsort.org) were used. The subcellular localization of a protein is underlined with the following letter code (c: cytoplasm; p: peroxisome, u: unknown, m&p: mitochondria and plasmatic membrane, v: vacuole) and the chromosomal location is the number of chromosome shown in brackets, domains of the protein are indicated (Cytb5-R: Cytochrome b5 reductase, u: unknown), partners indicate the need of other proteins for activity (Cyt-c: Cytochrome c, u: unknown).

Due to the reported cross-effect between molybdate, sulphate and phosphate transport in plant cells, it was thought that cellular needs of Mo were fulfilled by a non-specific molybdate supply via the plant sulphate or phosphate transporters.15,16 In tomato plants molybdate uptake has been shown to be enhanced by the presence of phosphate and decreased by sulphate.16 This statement was reinforced by the lack of specific molybdate transporters in plants. Early indications of the existence of specific molybdate transporters in plants were reported in the green alga Chlamydomonas reinhardtii suggesting the presence of at least two molybdate transport activities with different kinetic properties: one of them with high affinity and low capacity, and the other one with low affinity and high capacity.17 These physiological data were confirmed with the identification of the high affinity molybdate transporter family MOT1.18 Members of this family have been characterized and related to molybdate transport in C. reinhardtii and Arabidopsis.18–21 Subsequently a second type of molybdate transporter, MOT2, unrelated to the

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MOT1 family was identified and characterized in C. reinhardtii.22 None of the reported plant molybdate transporters has been related to the outward transport of Mo from the cell cytoplasm, therefore this process is still unknown. Mo toxicity cases are few in plants. Identification of proteins involved in Mo export would explain why plants are fairly tolerant to Mo. In C. reinhardtii, the deficiency in functionality of the molybdate transporter MOT1 (Nit5 mutant) was shown to confer resistance to high molybdate concentrations (50 mM).17 An efficient Mo storage mechanism could also contribute to Mo plant tolerance. Different mechanisms of Mo storage have been reported in plants. In C. reinhardtii Mo is stored in the form of Moco bound to the Molybdenum cofactor Carrier Protein (MCP).23,24 To date no homologues of MCP have been found in higher plants. Interestingly Arabidopsis has a family of nine proteins classified as lysine decarboxylase-like proteins (named Moco Binding Protein, MBP) showing significant structural similarity to C. reinhardtii MCP; however only four of these

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proteins are able to bind Moco with high KD values, suggesting that MBPs have a role different to Moco storage.25 In addition, levels of anthocyanin and malic acid seem to be directly related to molybdate accumulation in Brassica sp. and Medicago sativa L.26–28 These results point to a role of these compounds in Mo sequestration and storage in plants (Fig. 1).

MOT1 family

Fig. 1 Organization of the Mo homeostasis machinery in a single plant cell. Mo, as molybdate, enters in the cell by specific transporters belonging to the MOT1 or MOT2 families. In the cell Mo is stored in the vacuole or complexed by anthocyanin or malic acid. Mo coming either from the external medium or from the vacuole is used by the cell to form Moco via the catalytic activity of CNX1. MOT1;2 has been proposed to export Mo from the vacuole to the cytosol. The exact subcellular location of MOT1;1 is not clear since conflicting data suggest its location in the plasma membrane or in the mitochondria. Moco is used by Moenzymes to carry out its biological activity or is stored and protected against oxidation by MCP. Transporters represented with dashed lines have unclear or unknown subcellular location.

First a eukaryotic molybdate transporter was reported in parallel in C. reinhardtii and Arabidopsis as belonging to the MOT1 family (MOlybdate Transporter type 1).18,21 Members of this family of proteins are present in higher plants, algae, fungi and bacteria and are probably related to molybdate transport/ accumulation (Fig. 2a).18 C. reinhardtii MOT1 (CrMOT1;1) was identified on the basis of the above-mentioned relationship between sulphate and molybdate transport since MOT1 proteins are distantly related to the plant sulphate transporter family SULTR, but different enough to be considered as an independent family. The main differences between MOT1 and SULTR proteins are: (i) the lack of the STAS (Sulphate Transporter and Anti-Sigma antagonist) domain in proteins from the MOT1 family. This motif is present in members of the SULTR and has been demonstrated to be essential for sulphate transport activity;29,30 (ii) the presence of two conserved motifs in all

Fig. 2 Phylogenetic trees of plant MOT1 (a) and MOT2 (b) proteins. Alignment of the protein sequence was performed by the CLUSTAL method. Phylogenetic data were obtained by the Mega 5.1 software package. Colours represent different plant organisms; algae are coloured in blue, monocotyledons are coloured in green and dicotyledons are coloured in orange. Accession numbers (a): A. thaliana MOT1;1, NP_180139; A. thaliana MOT1;2, BAF01113; B. napus, CAC39421; C. reinhardtii, A6YCJ2; C. variabillis, EFN52559; H. vulgare, BAJ94535; L. japonicus, AFK43331; O. tauri, XP_003081530; O. sativa, NP_001043703; P. tadea, CV034997; P. trichocarpa, XP_002308631; R. communis, XP_002517550; S. bicolor, XP_002458231; V. carteri, XP_002951222; V. vinifera, XP_002281989; Z. mays, DAA54584. Accession numbers (b): A. thaliana, NP_567786; C. reinhardtii, AEY68285; C. variabilis, EFN51339; G. max, XP_003526731; M. trucatula, XP_003602305; O. sativa, NP_001048746; O. tauri, XP_003078107; P. tadea DR016373; P. sitchensis, ABR17540; P. trichocarpa, XP_002318449; R. communis, BAJ88961; S. bicolor, XP_002466934; H. vulgare, BAJ88961; V. carteri, XP_002948878; V. vinifera, XP_002280860; Z. mays, DAA42909.

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proteins belonging to the MOT1 family but not in proteins from the SULTR family.18 Functionality studies of CrMOT1 by antisense RNA showed that the silencing of CrMOT1 transcription is directly connected to the inhibition of molybdate uptake and reduction of the activity of the Mo-containing enzyme NR.18 Furthermore, direct molybdate transport measurements in C. reinhardtii revealed that the process mediated by CrMOT1 has a high affinity, with a Km value of 7 nM (Table 1).18 In addition competition studies carried out in Saccharomyces cerevisiae after over-expression of CrMOT1 showed inhibition by the molybdate-analogous anion tungstate but not by high concentration of sulphate, suggesting that the transport activity of CrMOT1 is specific for molybdate.18 The regulation profile of CrMOT1 is different from the already known bacterial molybdate transporter (ModABC). While ModABC is transcriptionally regulated by intracellular concentrations of molybdate,31 the transcription and activity of CrMOT1 are regulated by nitrate and insensitive to Mo availability.18 This regulation pattern points to a role of CrMOT1 in Mo supply when the cell needs to assimilate nitrate as a nitrogen source, and thus it requires a high activity of the Mo-containing enzyme NR. CrMOT1 activity could be a coordination point between Moco biosynthesis and nitrate assimilation in C. reinhardtii. In Arabidopsis two members of the MOT1 family (AtMOT1;1 and AtMOT1;2) have been identified. AtMOT1;1 was identified from a 3-fold decrease of Mo content in the accession Landsberg erecta with respect to the accession Columbia.21 Genetic analysis revealed that this alteration in Mo content is caused by differences in the gene previously annotated as Sultr5;2 and finally named AtMOT1;1. Functional characterization of AtMOT1;1 revealed a specific and high affinity molybdate transport process with a Km of 20 nM and insensitive to high amounts of sulphate in the medium.21 These characteristics are similar to those described above for CrMOT1. However, in contrast to CrMOT1, AtMOT1;1 presents a transcriptional profile regulated by molybdate. Shoots and roots of plants grown under Mo-limiting conditions present a reduced AtMOT1;1 expression.21 AtMOT1;1 seems to be essential for efficient uptake of molybdate from soil and it is not directly connected with nitrate assimilation. Localization of AtMOT1;1 within the cell is not clear since two different subcellular localizations, plasma membrane and mitochondria, have been reported for this protein.19,21 Although the plasma membrane is a more suitable localization for a transporter involved in molybdate uptake from soil, experimental and in silico data point to a mitochondrial location for AtMOT1;1 suggesting a role of this organelle in Mo homeostasis. However, further studies are needed to clarify cellular localization of AtMOT1;1 and thus its physiological role in molybdate transport/accumulation. A second member of the MOT1 family has been identified in Arabidopsis on the basis of its high sequence similarity to AtMOT1;1 (72%).20 This protein has been called AtMOT2; however this transport protein clearly belongs to the MOT1 family and in order to avoid any confusion with proteins belonging to the recently reported molybdate transporters

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family MOT2, we recommend to rename it as AtMOT1;2 (Arabidopsis thaliana MOlybdate Transporter type 1; member 2). Arabidopsis plants lacking functional AtMOT1;2 present an altered profile of Mo accumulation in plant tissues suggesting a role of this protein in interorgan allocation of molybdate.20 AtMOT1;2 shows a clear vacuolar localization pointing to the vacuole as a Mo storage compartment in plants. Notwithstanding there is no evidence linking AtMOT1;2 and Mo uptake/ accumulation; i.e. molybdate transport activity of this protein has not yet been confirmed. Further studies are needed to clarify AtMOT1;2 function within the overall Mo homeostasis mechanism in plants. Proteins of the MOT1 family are significantly conserved with CrMOT1 and are present in trees and herbaceous mono- and dicotyledonous plants, and algae, but not in animals (Fig. 2a).

MOT2 family In C. reinhardtii, at least two molybdate transport activities have been reported.17,18 However, only one MOT1 member is present in the genome of this alga.18 Therefore, the second molybdate transport activity present in C. reinhardtii is not related to the MOT1 family. On this basis a second transport activity, CrMOT2, was identified in C. reinhardtii.22 An RNA antisense strategy showed that molybdate uptake in Chlamydomonas depends on CrMOT2 transcription only in the absence of CrMOT1 activity.22 Molybdate transport mediated by CrMOT2 is also a high affinity process but characterized by a Km (550 nM) higher than the value reported for CrMOT1.22 Furthermore, transport competition studies using tungstate and sulphate suggest that CrMOT2 is a specific molybdate transporter.22 In contrast to CrMOT1, regulation of CrMOT2 is dependent on Mo availability, since CrMOT2 transcription is upregulated by Mo starvation,22 pointing to different roles of CrMOT1 and CrMOT2 within the Mo homeostasis mechanism in Chlamydomonas. While CrMOT1 seems to be connected to the NR activity, CrMOT2 could be essential under molybdate deficient conditions. Proteins showing significant similarity to CrMOT2 are present in trees and herbaceous mono- and di-cotyledonous plants, algae and animals (Fig. 2b). Heterologous expression in S. cerevisiae reveals that human MOT2 (HsMOT2) is able to transport molybdate from the external medium at nanomolar concentrations. This result suggests that HsMOT2 could also act as a molybdate transporter in humans.22 CrMOT2 is the only plant member of the MOT2 family whose functionality as a molybdate transporter has been experimentally demonstrated. Additional studies involving plant MOT2 proteins are required to verify its role in Mo transport/accumulation.

Other molybdate transport proteins in plants The high affinity sulphate transporter SHST1 has been shown to be involved in low affinity molybdate transport in the higher plant Stylosanthes hamata.32 SHST1 is able to enhance molybdate

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uptake at nanomolar concentrations after over-expression in S. cerevisiae even in the presence of sulphate in the medium. However sulphate transport via SHST1 is inhibited by the presence of molybdate.32 These findings support the cross-effect of molybdate and sulphate previously observed in plants at a physiological level.16 In addition a molybdate transport-defective mutant (DB6) has been identified in C. reinhardtii showing a low activity of the Mo-containing enzymes NR and XDH.33 This phenotype is partially repaired by adding 10 mM molybdate to the growth medium, pointing to a molybdate transport deficiency.33 Genetic complementation studies using the DB6 mutant showed that a wild type copy of CrMOT1 is not able to restore its deficiency in molybdate uptake. Further analyses are required to clarify whether DB6 is affected in CrMOT2, in a non-yet identified molybdate transporter gene or in an unknown molybdate uptake regulatory gene.

Activation of Mo to form molybdopterin In plants Moco is synthesized in an ancient and conserved pathway involving two cellular compartments, mitochondria and cytosol, and four biosynthetic steps. Prior to Moco incorporation into Mo enzymes, the Mo atom has to be activated in four steps. After incorporating synthesized Moco, some enzymes require one additional maturation step, the addition of terminal inorganic sulphur to the Mo-centre. The genes and proteins involved in plant Moco synthesis are named according to the CNX nomenclature (Cofactor for Nitrate reductase and Xanthine dehydrogenase).

The first step, GTP circularization The first step in Moco biosynthesis is called circularization and involves the conversion of GTP into a sulphur-free pyranopterin called cPMP (former precursor Z) that carries a geminal diol (Fig. 3).34 It is well established that each carbon of the GTP skeleton is incorporated into cPMP.35 This reaction is catalyzed in all eukaryotic organisms by two proteins: CNX2 a radical S-adenosylmethionine-dependent (SAM) enzyme and a hexameric protein called CNX3. CNX2 has two oxygen-sensitive [4Fe–4S] clusters, the N-terminal Fe–S cluster involved in the reductive cleavage of SAM that generates a 5 0 -deoxyadenosyl radical and the C-terminal Fe–S cluster crucial for substrate binding and/or activation.36 The precise function of CNX3 is not clear, but since some radical SAM enzymes require another protein onto which the radical is transferred, it is speculated that CNX3 might have a similar function.37 In plants CNX2 and CNX3 are encoded by different genes located in separate chromosomes; however in humans those genes are encoded as bicistronic with two consecutive ORFs that encode two enzymatic activities.38 The Arabidopsis39 and Chlamydomonas (personal observation) CNX2 and CNX3 proteins have N-terminal extensions carrying targeting signals. The Arabidopsis proteins have been localized in mitochondria.40 This makes sense because, for its functioning CNX2 needs Fe–S clusters which are readily available in the mitochondrion. Once cPMP is formed, Moco synthesis is completed in the cytosol.

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Fig. 3 Plant Moco biosynthetic pathway. The basic steps of Moco biosynthesis are shown. All known intermediates and the different CNX proteins of the pathway are presented sequentially in the four steps in which Moco is synthesized from GTP.

This means that cPMP has to pass through the mitochondrial membranes to the cytosol. This is carried out by the transporter protein ATM3, which is localized in the inner membrane of mitochondria and belongs to the family of ATP-binding cassette (ABC) transporters.40 The precise role of ATM3 is still unknown. These mitochondrial transporters are related to the maturation of cytosolic Fe–S proteins and to the resistance of heavy metals.41 Mutations in Arabidopsis ATM3 result in a 50% reduction of the activities of Moco-containing enzymes NR and SO, whereas the activities of XDH and AO also depending on Fe–S clusters are undetectable.40 Also human cells possess this transporter protein, which is named ABCB7.42

The second step, MPT synthesis In order to generate the dithiolene MPT two sulphur atoms are transferred to cPMP by the MPT synthase (Fig. 3). MPT synthase is a heterotetrameric complex of two small (CNX7) and two large subunits (CNX6) that stoichiometrically converts cPMP into MPT. A two-step mechanism for MPT dithiolate formation is required since each small subunit of MPT synthase carries a sulphur atom.43 The formation of MPT is not properly a catalytic reaction; therefore MPT synthase needs to be regenerated upon

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each reaction step. In order to reactivate the enzyme the MPT synthase is re-sulphurated by the MPT synthase sulphurase called CNX5, which contains a C-terminal rhodanese-like domain. Two highly conserved CXXC motifs are present in CNX5,44 which are involved in the binding of Zn2+.45 The crystal structure of the CNX5 prokaryotic homologue reveals that the Zn-binding site is very distant from the active site, which suggests a structural role of Zn.46 CNX5 is a two domain protein that activates CNX7 by adenylation (carried out by the CNX5 N-terminal domain) followed by a sulphur transfer reaction (carried out by the CNX5 C-terminal domain which has a rhodanese function). Cysteine is a likely candidate as a final S-donor for the formation of MPT.47,48 It has been demonstrated that the Arabidopsis CNX5 is strictly required for the thio-modification of cytosolic tRNAs; in vivo when expressed in Saccharomyces cerevisiae, CNX5 is able to substitute for the corresponding yeast orthologs in the thio-modification of yeast cytosolic tRNAs.49 These findings suggest that in plants CNX5 is involved in both the thio-modification of tRNA and MPT biosynthesis.

The third step, MPT activation After synthesis of the MPT moiety the Moco chemical backbone is ready to bind Mo. However the MPT moiety has to be activated by adenylation prior to chelation with Mo, leading to the formation of the intermediate MPT-AMP (Fig. 3).50 Most eukaryotes catalyze the MPT adenylation by two-domain proteins, such as Arabidopsis CNX1 (G domain and E domain); however prokaryotes use two independent proteins. The orientation of CNX1 domains is inverted in the Arabidopsis protein compared to the human protein (Gephyrin), reflecting separate evolutionary events.51 This points to a functional cooperation between their domains, as they combine functions of proteins that are separately expressed in E. coli. Recently, an in vitro Moco biosynthesis system has been used to compare the reaction rates of the individual Gephyrin domains with the full protein, suggesting a product–substrate channelling as the functional origin of domain fusion in Gephyrin and also in CNX1.52 Arabidopsis CNX1 is able to bind to the cytoskeleton,53 this anchoring might help to organize and to make efficient the Mo transfer to MPT. Chlamydomonas is one of the few eukaryotes, together with Caenorhabditis elegans, that present CNX1 encoded by two separate genes each responsible for the polypeptides CrCNX1G and CrCNX1E. Chimeric fusions of Chlamydomonas CNX1G and CNX1E in both orientations are able to restore efficiently the eukaryotic Moco biosynthesis.54 The Chlamydomonas CNX1E is a unique eukaryotic protein characterized as being able to restore in vivo the prokaryotic Moco biosynthesis.54 This means that the Chlamydomonas protein has retained the ability to interact with prokaryotic proteins; furthermore the lack of this ability in other eukaryotic proteins might be due to their domain fusions. The crystal structure of Arabidopsis CNX1G clarifies the kinetic mechanism of MPT adenylation.50 This adenylation is

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Mg2+ and ATP dependent and results in MPT-AMP bound to CNX1G.55 The crystal structure of CNX1G also reveals a Cu atom bound to the MPT dithiolate sulphurs.50 The real function of Cu during Moco biosynthesis is still unknown; it may participate in the sulphur-transfer reaction acting as a protecting group for MPT dithiolate sulphurs or improve the Mo insertion. In vitro studies with MPT-AMP bound to CNX1G reveal an inhibition of Moco synthesis in the presence of 1 mM CuCl2, therefore a competition between Cu and Mo during Moco biosynthesis has been suggested,50 raising the question of whether conditions of Cu overload or deficiency might affect Moco biosynthesis. Therefore, when studying Cu homeostasis the analysis of Mo metabolism could shed further light on the link between Mo and Cu competition since increased cellular Cu concentrations might be the cause for a decreased rate of Moco synthesis.

The fourth step, Mo insertion The final step, called Mo insertion, physically links molybdate to MPT. The active intermediate MPT-AMP is deadenylated after its transfer from CNX1G to CNX1E in a reaction that is molybdate and Zn2+/Mg2+-dependent (Fig. 3).56 It has been demonstrated that MPT-AMP and molybdate bind in a cooperative way to the CNX1E domain.56 Artificially added tungstate and sulphate are able to compete with molybdate in the binding to the CNX1E domain, which demonstrates the presence of an anion-binding site for molybdate in CNX1E. It has been proposed that once MPT-AMP and molybdate are bound to CNX1E, molybdate oxygen may promote pyrophosphate cleavage. This attack would result in the formation of adenylated molybdate (as a hypothetical reaction intermediate) and its subsequent use as a Mo source.56 However, the formation of adenylated molybdate has not been experimentally demonstrated, probably due to its transient nature. As MPT species not loaded with Cu were also converted into Moco, Cu is neither involved in MPT-AMP cleavage nor Mo insertion, therefore Cu would seem to act as a protecting group for MPT dithiolate rather than being involved in Mo insertion.56 In Chlamydomonas, the mutations nit4 and nit6 are the result of a single change in CNX1E (V171A and G183D, respectively) leading to its lack of function. The mutated CNX1E versions prevent the synthesis of active Moco and therefore lead to the inability to use nitrate as a nitrogen source.57 In addition, these CNX1E variants were able to complement each other intermolecularly, which is shown by both in vivo and in vitro studies. The intermolecular complementarity of CNX1E variants provides useful information about the roles of these particular residues in the CNX1E function.57 Upon its reaction, Moco is stoichiometrically released from CNX1E56 and subsequently assembled into apo-Mo-enzymes or bound to MCP.24

The sulphuration of Moco in XDH and AO After Moco insertion, in order to gain enzymatic activity the Mo hydroxylases such as AO and XDH require an additional step

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Tutorial Review called sulphuration consisting of the addition of terminal inorganic sulphur to the Mo-centre. That is catalyzed in plants by the Moco sulphurase ABA3 (abscisic acid deficient 3) a homodimer acting as two domain protein.58 In ABA3 the N-terminal NifS-like domain takes in a pyridoxal phosphate dependent manner sulphur from L-cysteine and forms a persulphide intermediate on a conserved cysteine residue (Cys430).59 The number of surface-exposed cysteine residues has been determined in ABA3 identifying a second persulfide-binding cysteine residue (Cys206).60 It is tempting to speculate that the persulfide is transmitted within the ABA3 protein from the initial persulfide-binding cysteine residue to one or several other cysteine residues before reaching its destination.60 The presence of these invariant cysteine residues led to propose a model in which that domain might be a sulphur carrier domain61 that receives sulphur and delivers it for the formation of sulphurated Moco. The C-terminal domain of ABA3 binds sulphurated Moco efficiently.62 Then, there appears the question of how the sulphurated Moco is transferred from ABA3 to AO or XDH. There are two possibilities: (i) the terminal sulphur is transferred as such to the Moco yet assembled in AO and XDH, or (ii) the whole sulphurated Moco molecule is transferred to AO and XDH apo-proteins. The second alternative must be seriously considered since the ABA3-bound cofactor can be removed without disturbing the protein integrity, and at the same time this cofactor-free ABA3 binds tightly the cofactor when provided exogenously.61 However, experimental data are needed to exclude or support one of both possibilities. Considering physiological aspects, as the plant AO is involved in the synthesis of the plant hormone abscisic acid, the sulphuration step may be a regulatory switch point controlling the amount of this hormone in the cell. The activity of ABA3 could control the amount of functional AO. In fact, a rapid induction of the aba3 messenger upon drought and salt stress in plants has been observed.63 The overexpression of the ABA3 gene increased the abscisic acid level and drought tolerance in transgenic cotton64 and tobacco65 plants. The overexpression of the Arabidopsis ABA3 gene in maize also markedly enhanced the expression of AO activity, leading to ABA accumulation and an increased drought tolerance.66 In many species AO and XDH are also involved in reactive oxygen species (ROS) formation. In tomato and Arabidopsis XDH and AO are capable of producing ROS, and this production is dependent on Moco sulphuration.67 Plant ROS production and transcript levels of AO and XDH are rapidly upregulated by application of abscisic acid and in water-stressed leaves and roots. These results support that plant ABA3 is possibly a novel source for ROS regulation.

Moco-containing enzymes Five Moco-containing enzymes exist in plants: NR, SO, XDH, AO and ARC (see Table 2).14,68,69 NR is an enzyme essential for utilization of nitrate as a nutrient, which in addition has a signaling role in plant differentiation.70,71 NR is located in the cytosol as a homodimer, each monomer of about 105 kDa

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Metallomics contains three prosthetic groups: Moco in the N-terminal domain, cytb5/heme in a central domain and FAD at the C-terminus, each linked by two short hinge regions, one of which confers posttranslational regulatory properties by the binding of 14-3-3 proteins.72 Though this domain structure is well conserved in plants, sequences in the hinge region between Moco and cytb5/heme domains, (K/R)(S/T)XS*(T/S)XP, are absent in mosses and algae.73,74 This sequence is the target of phosphorylation and binding of 14-3-3 proteins that down regulates the enzyme activity by inducing a conformational change that increases the distance between two redox-active sites.75 This regulatory mechanism common to vascular plants is absent in bryophytes and algae. NAD(P)H is the electron donor for nitrate reduction to nitrite.71,76 In addition, NR might also use nitrite as a substrate producing the oxygen reactive species nitric oxide (NO), an important signaling molecule in plants.77,78 Though the in vivo activity of NR for NO formation is approximately 1% of the enzyme activity for nitrite production79 the role of this enzyme in NO production in plants has been highlighted.80 SO participates in the elimination of toxic sulphite by its oxidation to sulphate. Sulphite is produced in plants during the sulphur degradative metabolism, and sulphate is reassimilated in the chloroplast after its conversion to 3 0 -phosphoadenosine 5 0 -phosphosulphate (PAPS).81 SO from Arabidopsis is a homodimer with 45 kDa subunits, located in the peroxisome, which uses oxygen as the electron acceptor producing sulphate and hydrogen peroxide.82 However, SO from the green alga Chlamydomonas is an animal-type enzyme protein with mitochondrial localization: a homodimer of 55 kDa subunits each containing an N-terminal cytochrome b5 (cyt b5)-binding domain and a C-terminal Moco-binding domain.83 This protein increases its expression during sulphur deprivation, since the degradative metabolism of S-containing metabolites is stimulated under these conditions.84 Two highly homologous and tandemly duplicated XDH genes are present in the Arabidopsis genome (Table 2). AtXDH1 is responsible for most of the XDH activity in the plant and is overexpressed under stress conditions, while AtXDH2 is constitutively expressed.85 AtXDHs are homodimers with 150 kDasubunits that contain the N-terminal domain binding Fe–S cluster of the Fe2S2 type followed by an FAD/NAD binding domain, and a C-terminal Moco binding domain. AtXDH1 uses xanthine and hypoxanthine as main substrates and strictly NAD+, but is able to be a potent producer of superoxide anions through its NADH oxidase activity.86 Though XDHs are cytosolic enzymes, plant peroxisomes contain xanthine, uric acid and XDH in both inter-convertible forms, dehydrogenase and oxidase.87,88 Thus, it appears that XDH proteins would show particular characteristics depending on their subcellular localization. In Chlamydomonas, an inducible XDH structurally similar to the plant enzyme allows the use of xanthine and hypoxanthine as a sole nitrogen source.89 The algal genome contains a single XDH gene that is upregulated under nitrogen deficiency conditions.90

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Aldehyde oxidases seem to be highly represented mostly in multicellular organisms.91 In fact four AO genes (AAO1-4) are present in Arabidopsis (Table 2). They seem to be the result of duplication events from an ancestral XDH gene. It is interesting to point out that the fungus Aspergillus nidulans has a single locus encoding XDH,92 whereas Chlamydomonas has an additional gene probably encoding an AO protein together with the XDH gene (Table 2).93

The amidoxime reducing complex (ARCO) Recently a new Moco-containing enzyme has been identified in eukaryotic69 and prokaryotic94 organisms. This protein seems to be involved in the reduction of a broad range of N-hydroxylated compounds (NHC). Interestingly, these Moco proteins need other partner proteins which donate the reducing power necessary for the NHC reductions. The first evidence about the presence of a Moco-enzyme involved in the NHC reductions was reported in studies about the toxicity of the base analogs 6-hydroxylaminopurine (HAP) in bacteria,95 a powerful mutagen; although the molecular identity of the Moco-enzyme was not deciphered.96 Further studies in humans revealed the existence of a Moco-protein able to convert several amidoxime prodrugs into their active amino forms; this Moco-protein was

called ARC (Amidoxime Reducing Component).69 This conversion is analogous to the conversion of HAP to adenine, as it involves the reduction of an NHC to the corresponding amino form. In humans there are two ARC proteins (hmARC1 and hmARC2) taking part in a three-component system consisting of the ARC enzyme, cytochrome b5 and NADH cytochrome b5 reductase.69 Therefore we propose to call the complex formed between ARC and their partners as ARCO (Amidoxime Reducing COmplex). Interestingly, the bacterial ARCO is a two-component system that instead of cytochrome uses ferredoxin94 fused to the ARC enzyme in the C-termini (YcbX) and as a second component uses a flavin reductase instead of NADH cytochrome b5 reductase (CysJ) (Fig. 4).97 In Chlamydomonas, ARCO consists of the Moco-enzyme CrARC, cytochrome b5 and NADH cytochrome b5 reductase similarly to its human counterpart and differently from the prokaryotic system (Fig. 4). The Chlamydomonas ARCO shows a Zn2+-dependent activity, the presence of Zn2+ increases its Vmax more than 14-fold.68 CrARC belongs to the sulphite oxidase family since the Cys252 residue has been identified as a putative ligand of the Mo atom (Fig. 4).68 However, in humans the reconstitution of active Moco onto recombinant ARC in the absence of sulphur indicates that it belongs neither to the xanthine oxidase family, nor to the sulphite oxidase family since none of its nine cysteine residues could be identified as a putative ligand of

Fig. 4 Phylogenetic tree of plant ARC proteins. Alignment of the protein sequences was performed by the CLUSTAL method. Phylogenetic data were obtained by the Mega 5.1 software package. Colours represent different plant organisms; algae are in blue, monocotyledons are in green and dicotyledons are in orange. Accession numbers: A. thaliana 1, NP_174376.1; A. thaliana 2, NP_199285.1, M. truncatula 1, XP_003594858.1; M. truncatula 2, XP_003594859.1; M. truncatula 3, XP_003594860.1; Z. mays, NP_001148545; O. sativa, NP_001063929; V. vinifera, XP_002273557.1; G. max, XP_003533626; S. bicolour, XP_002460665.1; B. distachyon, XP_003578651.1; P. trichocarpa, ABK95960; R. communis 1, XP_002528142; R. communis 2, XP_002528139; L. japonicas, AFK45042.1; C. reinhardtii, AEI61922; C. variabillis, EFN55110; O. lucimarinus, XP_001421346; V. carteri, XP_002954725.

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Tutorial Review the Mo atom. Therefore, the Mo coordination sphere of the ARC proteins in humans is yet unknown.98 Arabidopsis, as many eukaryotes, also contains two ARC versions with both showing strong similarities at amino acid and nucleotide levels, however Chlamydomonas has only one (Fig. 5).99 The plant ARC family is significantly conserved with CrARC and its members are present in trees, herbaceous monoand di-cotyledonous plants and algae (Fig. 5). As there are unsequenced regions in the Chlamydomonas genome, the possibility of a second CrARC cannot be excluded. The plant ARC proteins contain two conserved domains: an N-terminal bbarrel domain and a MOSC domain. These domains were first found in eukaryotic Moco sulphurases (the abbreviation MOSC was introduced by Anantharaman and Aravind61 and refers to the Moco Sulphurase C-terminal domain). Moco sulphurases transfer inorganic sulphur from cysteine to the Mo centre of Moco as part of a final Moco maturation (sulphuration) step. Additional, MOSC-containing proteins are distributed widely in pro- and eukaryotes; however, except for the Moco sulphurases, all other members of the MOSC superfamily are proteins without any confirmed function.61 The ARC proteins show several conserved patches of hydrophobic residues and an absolutely conserved cysteine residue located in the C-terminal region of the domain that could be considered part of their signature. This cysteine is reminiscent of the analogous conservation of a cysteine in the active site of the thioredoxin and rhodanese superfamilies.61 Members of these superfamilies have been implicated in the synthesis of Fe–S clusters. These observations suggest that the MOSC domain might play a critical role in the formation of diverse metal–sulphur clusters, with the

Metallomics conserved cysteine playing an active role in this process. The N-terminal b-barrel domain that is undetectable elsewhere in standalone form is predicted to build a b-strand-rich fold like structure. This particular domain may have specific roles in interaction with substrates of these enzymes. The subcellular localization of ARC proteins is not well defined. In humans the ARC protein is associated with the outer mitochondrial membrane.69,100 Mouse ARC proteins were localized in the inner mitochondrial membrane101 and rat ARC proteins were localized in peroxisomal membranes.102 However, the Arabidopsis and Chlamydomonas counterparts lack a clear targeting signal for organellar export.99 The physiological role of ARCO is at present not known. However, taking into account its activity of conversion of HAP67 and N-hydroxy-cytosine,98 a role in preventing the accumulation of mutagenic base analogues in the cell could be proposed.103 Cellular DNA is protected from misincorporation of toxic N-hydroxylated base analogues during replication by converting them to the correct purine or pyrimidine bases. In addition, in vitro studies have recently shown that human ARCO is able to catalyze the reduction of the NO precursor N4-hydroxy-L-arginine (NOHA) to arginine.104 Whether ARCO is involved in physiological NOHA reduction and/or is capable of physiologically affecting the NO levels has to be supported by future experiments. Interestingly the down-regulation of ARCO resulted in a significant decrease of the intracellular lipid levels.105 However, the exact role of ARCO in lipid synthesis requires further research. In plants, it has been shown that the Chlamydomonas strains defective in Moco biosynthetic genes are sensitive to high content of NHC in the growth media.68 The generation of an ARCO silencing/knockdown in plant-type organisms together with the study of ARCO overexpression should be addressed to further elucidate the potential physiological function of this protein complex. Interestingly the recombinant expression of ARC proteins reveals that they are monomeric,98 in contrast to all other eukaryotic Moco enzymes that act as dimers. The crystal structure of ARC is still unknown and its future resolution will improve our understanding of its particular function.

Conclusions and perspectives

Fig. 5 The amidoxime reducing complex (ARCO). The figure shows: (A) the structure of the Moco molecule illustrating that the fifth Mo ligand in CrARC is the cysteine 252. (B) Schematically the ARCO in E. coli and (C) in Chlamydomonas. In colors, each of the protein domains found in the ARCO; MOSC (Molybdenum Cofactor Sulfurase C-terminal domain), Cyt b5 (Cyt b5 domain), FAD/NADH (FAD and NADH binding domains), Fe–S (2Fe-2S binding domain) and FMN (FMN binding domain). The MOSC domain is able to bind Moco, which is indicated by Moco above the domain. In the bottom it is illustrated that for the reductions of an N-hydroxylated compounds the electron donor in the Chlamydomonas system is NADH and in the E. coli system NADPH.

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Because of the conservation of processes using the scarce element Mo and its active form of Moco, many new aspects learnt in a system can be extrapolated to others, of course, with adequate cautions. In spite of having identified high affinity molybdate transporters, we are far from a detailed understanding of Mo homeostasis since important points are still lacking. Among others we can ask ourselves different questions such as: is there a molybdate transporter to export the metal for balancing intracellular concentrations and for sending it to different plant tissues? What are their subcellular localizations and their redundancies within the cell and tissues? How is the intracellular traffic of molybdopterin and Moco? How does the transfer of Moco to the different molybdoenzymes occur? What is the functionality of the well-conserved ARCO system in plants?

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Abbreviations ABA3 AO Arabidopsis ARC ARCO ATM Chlamydomonas CNX cPMP Cyt b5 Fe–S GTP HAP mARC MBP MCP Moco MOSC MOT1 MOT2 MPT MPT-AMP NO NOHA NR PAPS PLP SO STAS XDH

Arabidopsis Moco sulfurase Aldehyde oxidase Arabidopsis thaliana Amidoxime reducing component Amidoxime reducing complex ABC transporter of the mitochondria Chlamydomonas reinhardtii Cofactor for nitrate reductase and xanthine dehydrogenase Cyclic pyranopterin monophosphate Cytochrome b5 Iron sulfur cluster Guanine triphosphate 6-Hydroxylaminopurine Mitochondrial amidoxime reducing component Moco binding proteins Moco carrier protein Molybdenum cofactor Moco-binding domain of eukaryotic sulfurases Molybdate transporter of high affinity type 1 Molybdate transporter of high affinity type 2 Molybdopterin Adenylated molybdopterin Nitric oxide N4-hydroxy-L-arginine Nitrate reductase 3 0 -Phosphoadenosine 5 0 -phosphosulfate Pyridoxal phosphate Sulfite oxidase Sulfate transporter and anti-sigma factor antagonist Xanthine oxidase

Acknowledgements ń This work was supported by Ministerio de Ciencia e Innovacio (MICINN) (Grant BFU2011-29338); European FEDER program; Junta de Andalucıá, Spain (PAI, BIO-128) and Universidad ´rdoba. We thank Maria Isabel Macias for technical de Co assistance.

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