RESUMO
Molybdenum (Mo) is vital for the activity of a small but essential group of enzymes called molybdoenzymes. So far, specifically five molybdoenzymes have been discovered in eukaryotes: nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and mARC. In order to become biologically active, Mo must be chelated to a pterin, forming the so-called Mo cofactor (Moco). Deficiency or mutation in any of the genes involved in Moco biosynthesis results in the simultaneous loss of activity of all molybdoenzymes, fully or partially preventing the normal development of the affected organism. To prevent this, the different mechanisms involved in Mo homeostasis must be finely regulated. Chlamydomonas reinhardtii is a unicellular, photosynthetic, eukaryotic microalga that has produced fundamental advances in key steps of Mo homeostasis over the last 30 years, which have been extrapolated to higher organisms, both plants and animals. These advances include the identification of the first two molybdate transporters in eukaryotes (MOT1 and MOT2), the characterization of key genes in Moco biosynthesis, the identification of the first enzyme that protects and transfers Moco (MCP1), the first characterization of mARC in plants, and the discovery of the crucial role of the nitrate reductase-mARC complex in plant nitric oxide production. This review aims to provide a comprehensive summary of the progress achieved in using C. reinhardtii as a model organism in Mo homeostasis and to propose how this microalga can continue improving with the advancements in this field in the future.
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Microalgae are used in various biotechnological processes, such as biofuel production due to their high biomass yields, agriculture as biofertilizers, production of high-value-added products, decontamination of wastewater, or as biological models for carbon sequestration. The number of these biotechnological applications is increasing, and as such, any advances that contribute to reducing costs and increasing economic profitability can have a significant impact. Nitrogen fixing organisms, often called diazotroph, also have great biotechnological potential, mainly in agriculture as an alternative to chemical fertilizers. Microbial consortia typically perform more complex tasks than monocultures and can execute functions that are challenging or even impossible for individual strains or species. Interestingly, microalgae and diazotrophic organisms are capable to embrace different types of symbiotic associations. Certain corals and lichens exhibit this symbiotic relationship in nature, which enhances their fitness. However, this relationship can also be artificially created in laboratory conditions with the objective of enhancing some of the biotechnological processes that each organism carries out independently. As a result, the utilization of microalgae and diazotrophic organisms in consortia is garnering significant interest as a potential alternative for reducing production costs and increasing yields of microalgae biomass, as well as for producing derived products and serving biotechnological purposes. This review makes an effort to examine the associations of microalgae and diazotrophic organisms, with the aim of highlighting the potential of these associations in improving various biotechnological processes.
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The stability and harmony of ecological niches rely on intricate interactions between their members. During evolution, organisms have developed the ability to thrive in different environments, taking advantage of each other. Among these organisms, microalgae are a highly diverse and widely distributed group of major primary producers whose interactions with other organisms play essential roles in their habitats. Understanding the basis of these interactions is crucial to control and exploit these communities for ecological and biotechnological applications. The green microalga Chlamydomonas reinhardtii, a well-established model, is emerging as a model organism for studying a wide variety of microbial interactions with ecological and economic significance. In this review, we unite and discuss current knowledge that points to C. reinhardtii as a model organism for studying microbial interactions.
RESUMO
Nitrogen (N) is an essential constituent of all living organisms and the main limiting macronutrient. Even when dinitrogen gas is the most abundant form of N, it can only be used by fixing bacteria but is inaccessible to most organisms, algae among them. Algae preferentially use ammonium (NH4+) and nitrate (NO3-) for growth, and the reactions for their conversion into amino acids (N assimilation) constitute an important part of the nitrogen cycle by primary producers. Recently, it was claimed that algae are also involved in denitrification, because of the production of nitric oxide (NO), a signal molecule, which is also a substrate of NO reductases to produce nitrous oxide (N2O), a potent greenhouse gas. This review is focused on the microalga Chlamydomonas reinhardtii as an algal model and its participation in different reactions of the N cycle. Emphasis will be paid to new actors, such as putative genes involved in NO and N2O production and their occurrence in other algae genomes. Furthermore, algae/bacteria mutualism will be considered in terms of expanding the N cycle to ammonification and N fixation, which are based on the exchange of carbon and nitrogen between the two organisms.
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Iron is an essential cofactor for symbiotic nitrogen fixation, required by many of the enzymes involved, including signal transduction proteins, O2 homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen-fixing nodule cells. Ferroportin family members in model legume Medicago truncatula were identified and their expression was determined. Yeast complementation assays, immunolocalization, characterization of a tnt1 insertional mutant line, and synchrotron-based X-ray fluorescence assays were carried out in the nodule-specific M. truncatula ferroportin Medicago truncatula nodule-specific gene Ferroportin2 (MtFPN2) is an iron-efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature and in inner nodule tissues, as well as in the symbiosome membranes in the interzone and early-fixation zone of the nodules. Loss-of-function of MtFPN2 alters iron distribution and speciation in nodules, reducing nitrogenase activity and biomass production. Using promoters with different tissular activity to drive MtFPN2 expression in MtFPN2 mutants, we determined that expression in the inner nodule tissues is sufficient to restore the phenotype, while confining MtFPN2 expression to the vasculature did not improve the mutant phenotype. These data indicate that MtFPN2 plays a primary role in iron delivery to nitrogen-fixing bacteroids in M. truncatula nodules.
Assuntos
Medicago truncatula , Regulação da Expressão Gênica de Plantas , Ferro/metabolismo , Medicago truncatula/genética , Medicago truncatula/metabolismo , Fixação de Nitrogênio , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , SimbioseRESUMO
The conservation of orthologs of most subunits of the origin recognition complex (ORC) has served to propose that the whole complex is common to all eukaryotes. However, various uncertainties have arisen concerning ORC subunit composition in a variety of lineages. Also, it is unclear whether the ancestral diversification of ORC in eukaryotes was accompanied by the neofunctionalization of some subunits, for example, role of ORC1 in centriole homeostasis. We have addressed these questions by reconstructing the distribution and evolutionary history of ORC1-5/CDC6 in a taxon-rich eukaryotic data set. First, we identified ORC subunits previously undetected in divergent lineages, which allowed us to propose a series of parsimonious scenarios for the origin of this multiprotein complex. Contrary to previous expectations, we found a global tendency in eukaryotes to increase or decrease the number of subunits as a consequence of genome duplications or streamlining, respectively. Interestingly, parasites show significantly lower number of subunits than free-living eukaryotes, especially those with the lowest genome size and gene content metrics. We also investigated the evolutionary origin of the ORC1 role in centriole homeostasis mediated by the PACT region in human cells. In particular, we tested the consequences of reducing ORC1 levels in the centriole-containing green alga Chlamydomonas reinhardtii. We found that the proportion of centrioles to flagella and nuclei was not dramatically affected. This, together with the PACT region not being significantly more conserved in centriole-bearing eukaryotes, supports the notion that this neofunctionalization of ORC1 would be a recent acquisition rather than an ancestral eukaryotic feature.
Assuntos
Complexo de Reconhecimento de Origem/metabolismo , Animais , Replicação do DNA/genética , Replicação do DNA/fisiologia , Eucariotos , Células Eucarióticas/metabolismo , Evolução Molecular , Duplicação Gênica/genética , Duplicação Gênica/fisiologia , Genoma/genética , Humanos , Imuno-Histoquímica , Complexo de Reconhecimento de Origem/genética , Filogenia , Ligação Proteica/genética , Ligação Proteica/fisiologiaRESUMO
Nitric oxide is a gaseous secondary messenger that is critical for proper cell signaling and plant survival when exposed to stress. Nitric oxide (NO) synthesis in plants, under standard phototrophic oxygenic conditions, has long been a very controversial issue. A few algal strains contain NO synthase (NOS), which appears to be absent in all other algae and land plants. The experimental data have led to the hypothesis that molybdoenzyme nitrate reductase (NR) is the main enzyme responsible for NO production in most plants. Recently, NR was found to be a necessary partner in a dual system that also includes another molybdoenzyme, which was renamed NO-forming nitrite reductase (NOFNiR). This enzyme produces NO independently of the molybdenum center of NR and depends on the NR electron transport chain from NAD(P)H to heme. Under the circumstances in which NR is not present or active, the existence of another NO-forming system that is similar to the NOS system would account for NO production and NO effects. PII protein, which senses and integrates the signals of the Câ»N balance in the cell, likely has an important role in organizing cell responses. Here, we critically analyze these topics.
RESUMO
Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron-molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. Medicago truncatula Molybdate Transporter (MtMOT) 1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. Loss-of-function mot1.2-1 mutant showed reduced growth compared with wild-type plants when nitrogen fixation was required but not when nitrogen was provided as nitrate. While no effect on molybdenum-dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen-fixing nodules, since genetic complementation with a wild-type MtMOT1.2 gene or molybdate-fortification of the nutrient solution, both restored wild-type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.
Assuntos
Proteínas de Transporte de Ânions/fisiologia , Medicago truncatula/metabolismo , Molibdênio/metabolismo , Proteínas de Plantas/fisiologia , Nódulos Radiculares de Plantas/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Medicago truncatula/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/ultraestruturaRESUMO
Symbiotic nitrogen fixation carried out by the interaction between legumes and diazotrophic bacteria known as rhizobia requires relatively large levels of transition metals. These elements are cofactors of many key enzymes involved in this process. Metallic micronutrients are obtained from soil by the roots and directed to sink organs by the vasculature, in a process mediated by a number of metal transporters and small organic molecules that facilitate metal delivery in the plant fluids. Among the later, nicotianamine is one of the most important. Synthesized by nicotianamine synthases (NAS), this molecule forms metal complexes participating in intracellular metal homeostasis and long-distance metal trafficking. Here we characterized the NAS2 gene from model legume Medicago truncatula. MtNAS2 is located in the root vasculature and in all nodule tissues in the infection and fixation zones. Symbiotic nitrogen fixation requires of MtNAS2 function, as indicated by the loss of nitrogenase activity in the insertional mutant nas2-1, phenotype reverted by reintroduction of a wild-type copy of MtNAS2. This would result from the altered iron distribution in nas2-1 nodules shown with X-ray fluorescence. Moreover, iron speciation is also affected in these nodules. These data suggest a role of nicotianamine in iron delivery for symbiotic nitrogen fixation.
RESUMO
All eukaryotic molybdenum (Mo) enzymes contain in their active site a Mo Cofactor (Moco), which is formed by a tricyclic pyranopterin with a dithiolene chelating the Mo atom. Here, the eukaryotic Moco biosynthetic pathway and the eukaryotic Moco enzymes are overviewed, including nitrate reductase (NR), sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase, and the last one discovered, the moonlighting enzyme mitochondrial Amidoxime Reducing Component (mARC). The mARC enzymes catalyze the reduction of hydroxylated compounds, mostly N-hydroxylated (NHC), but as well of nitrite to nitric oxide, a second messenger. mARC shows a broad spectrum of NHC as substrates, some are prodrugs containing an amidoxime structure, some are mutagens, such as 6-hydroxylaminepurine and some others, which most probably will be discovered soon. Interestingly, all known mARC need the reducing power supplied by different partners. For the NHC reduction, mARC uses cytochrome b5 and cytochrome b5 reductase, however for the nitrite reduction, plant mARC uses NR. Despite the functional importance of mARC enzymatic reactions, the structural mechanism of its Moco-mediated catalysis is starting to be revealed. We propose and compare the mARC catalytic mechanism of nitrite versus NHC reduction. By using the recently resolved structure of a prokaryotic MOSC enzyme, from the mARC protein family, we have modeled an in silico three-dimensional structure of a eukaryotic homologue.
Assuntos
Coenzimas/metabolismo , Enzimas/metabolismo , Metaloproteínas/metabolismo , Pteridinas/metabolismo , Animais , Miosinas Cardíacas/metabolismo , Coenzimas/biossíntese , Enzimas/química , Enzimas/genética , Células Eucarióticas/metabolismo , Mamíferos , Redes e Vias Metabólicas , Metaloproteínas/biossíntese , Molibdênio/metabolismo , Cofatores de Molibdênio , Cadeias Leves de Miosina/metabolismo , Nitrato Redutase/metabolismo , Nitritos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
Iron (Fe) is an essential micronutrient for symbiotic nitrogen fixation in legume nodules, where it is required for the activity of bacterial nitrogenase, plant leghemoglobin, respiratory oxidases, and other Fe proteins in both organisms. Fe solubility and transport within and between plant tissues is facilitated by organic chelators, such as nicotianamine and citrate. We have characterized a nodule-specific citrate transporter of the multidrug and toxic compound extrusion family, MtMATE67 of Medicago truncatula The MtMATE67 gene was induced early during nodule development and expressed primarily in the invasion zone of mature nodules. The MtMATE67 protein was localized to the plasma membrane of nodule cells and also the symbiosome membrane surrounding bacteroids in infected cells. In oocytes, MtMATE67 transported citrate out of cells in an Fe-activated manner. Loss of MtMATE67 gene function resulted in accumulation of Fe in the apoplasm of nodule cells and a substantial decrease in symbiotic nitrogen fixation and plant growth. Taken together, the results point to a primary role of MtMATE67 in citrate efflux from nodule cells in response to an Fe signal. This efflux is necessary to ensure Fe(III) solubility and mobility in the apoplasm and uptake into nodule cells. Likewise, MtMATE67-mediated citrate transport into the symbiosome space would increase the solubility and availability of Fe(III) for rhizobial bacteroids.
Assuntos
Ferro/metabolismo , Medicago truncatula/fisiologia , Fixação de Nitrogênio/fisiologia , Proteínas de Plantas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citratos/metabolismo , Regulação da Expressão Gênica de Plantas , Ferro/farmacocinética , Medicago truncatula/microbiologia , Mutação , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Simbiose/fisiologiaRESUMO
Molybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters. Members of the molybdate transporter family Molybdate Transporter type 1 (MOT1) were identified in the model legume Medicago truncatula and their expression in nodules was determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of a Transposable Element from Nicotiana tabacum (Tnt1) insertional mutant line were carried out in the one M. truncatula MOT1 family member specifically expressed in nodules. Among the five MOT1 members present in the M. truncatula genome, MtMOT1.3 is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. A mot1.3-1 knockout mutant showed impaired growth concomitant with a reduction of nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore, mot1.3-1 plants transformed with a functional copy of MtMOT1.3 showed a wild-type-like phenotype. These data are consistent with a model in which MtMOT1.3 is responsible for introducing molybdate into nodule cells, which is later used to synthesize functional nitrogenase.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Medicago truncatula/metabolismo , Molibdênio/metabolismo , Nitrogenase/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Zinc is a micronutrient required for symbiotic nitrogen fixation. It has been proposed that in model legume Medicago truncatula, zinc is delivered by the root vasculature into the nodule and released in the infection/differentiation zone. There, transporters must introduce this element into rhizobia-infected cells to metallate the apoproteins that use zinc as a cofactor. MtZIP6 (Medtr4g083570) is an M. truncatula Zinc-Iron Permease (ZIP) that is expressed only in roots and nodules, with the highest expression levels in the infection/differentiation zone. Immunolocalization studies indicate that it is located in the plasma membrane of nodule rhizobia-infected cells. Down-regulating MtZIP6 expression levels with RNAi does not result in any strong phenotype when plants are fed mineral nitrogen. However, these plants displayed severe growth defects when they depended on nitrogen fixed by their nodules, losing of 80% of their nitrogenase activity. The reduction of this activity was likely an indirect effect of zinc being retained in the infection/differentiation zone and not reaching the cytosol of rhizobia-infected cells. These data are consistent with a model in which MtZIP6 would be responsible for zinc uptake by rhizobia-infected nodule cells in the infection/differentiation zone.
Assuntos
Medicago truncatula/enzimologia , Medicago truncatula/microbiologia , Proteínas de Plantas/metabolismo , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/citologia , Nódulos Radiculares de Plantas/enzimologia , Zinco/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Homeostase , Medicago truncatula/genética , Modelos Biológicos , Fenótipo , Proteínas de Plantas/genética , Interferência de RNA , Nódulos Radiculares de Plantas/genética , Frações Subcelulares/metabolismoRESUMO
Molybdenum (Mo) is present in the active center of eukaryotic enzymes as a tricyclic pyranopterin chelate compound forming the Mo Cofactor (Moco). Four Moco containing enzymes are known in eukaryotes, nitrate reductase (NR), sulfite oxidase (SO), xanthine oxidoreductase (XOR), and aldehyde oxidase (AO). A fifth Moco enzyme has been recently identified. Because of the ability of this enzyme to convert by reduction several amidoximes prodrugs into their active amino forms, it was named mARC (mitochondrial Amidoxime Reducing Component). This enzyme is also able to catalyze the reduction of a broad range of N-hydroxylated compounds (NHC) as the base analogue 6-hydroxylaminopurine (HAP), as well as nitrite to nitric oxide (NO). All the mARC proteins need reducing power that is supplied by other proteins. The human and plants mARC proteins require a Cytochrome b5 (Cytb5) and a Cytochrome b5 reductase (Cytb5-R) to form an electron transfer chain from NADH to the NHC. Recently, plant mARC proteins were shown to be implicated in the reduction of nitrite to NO, and it was proposed that the electrons required for the reaction were supplied by NR instead of Cytochrome b5 components. This newly characterized mARC activity was termed NO Forming Nitrite Reductase (NOFNiR). Moonlighting proteins form a special class of multifunctional enzymes that can perform more than one function; if the extra function is not physiologically relevant, they are called promiscuous enzymes. In this review, we summarize the current knowledge on the mARC protein, and we propose that mARC is a new moonlighting enzyme. © 2017 BioFactors, 43(4):486-494, 2017.
Assuntos
Coenzimas/metabolismo , Metaloproteínas/metabolismo , Pteridinas/metabolismo , Aldeído Oxidase/metabolismo , Animais , Citocromos b5/metabolismo , Humanos , Cofatores de Molibdênio , Nitrato Redutase/metabolismo , Sulfito Oxidase/metabolismo , Xantina Desidrogenase/metabolismoRESUMO
Transition metals such as iron, copper, zinc, or molybdenum are essential nutrients for plants. These elements are involved in almost every biological process, including photosynthesis, tolerance to biotic and abiotic stress, or symbiotic nitrogen fixation. However, plants often grow in soils with limiting metallic oligonutrient bioavailability. Consequently, to ensure the proper metal levels, plants have developed a complex metal uptake and distribution system, that not only involves the plant itself, but also its associated microorganisms. These microorganisms can simply increase metal solubility in soils and making them more accessible to the host plant, as well as induce the plant metal deficiency response, or directly deliver transition elements to cortical cells. Other, instead of providing metals, can act as metal sinks, such as endosymbiotic rhizobia in legume nodules that requires relatively large amounts to carry out nitrogen fixation. In this review, we propose to do an overview of metal transport mechanisms in the plant-microbe system, emphasizing the role of arbuscular mycorrhizal fungi and endosymbiotic rhizobia.
RESUMO
Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Ferro/metabolismo , Medicago truncatula/metabolismo , Medicago truncatula/microbiologia , Proteínas de Plantas/metabolismo , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/microbiologia , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte de Cátions/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Técnicas de Inativação de Genes , Teste de Complementação Genética , Ferro/farmacologia , Manganês/metabolismo , Medicago truncatula/genética , Modelos Biológicos , Família Multigênica , Mutagênese Insercional/genética , Nitrogenase/metabolismo , Fenótipo , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rhizobium/efeitos dos fármacos , Nódulos Radiculares de Plantas/efeitos dos fármacos , Nódulos Radiculares de Plantas/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Simbiose/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
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 and S metabolism, hormone biosynthesis, toxic compound transformations and other important processes not only in plants but also in all the other kingdoms of life. Molybdate metabolism in plants is reviewed here, with special attention to two main aspects, the different molybdate transporters that with a very high affinity participate in molybdenum acquisition and the recently discovered Moco enzyme amidoxime-reducing component. Their functionality is starting to be understood.
Assuntos
Homeostase , Proteínas de Membrana Transportadoras/metabolismo , Molibdênio/metabolismo , Plantas/metabolismo , Proteínas de Transporte de Ânions/classificação , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/classificação , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Filogenia , Plantas/genéticaRESUMO
Molybdenum (Mo) is a very scarce element whose function is fundamental in living beings within the active site of Mo-oxidoreductases, playing key roles in the metabolism of N, S, purines, hormone biosynthesis, transformation of drugs and xenobiotics, etc. In eukaryotes, each step from Mo acquisition until its incorporation into a biologically active molybdenum cofactor (Moco) together with the assembly of this Moco in Mo-enzymes is almost understood. The deficiency in function of a particular molybdoenzyme can be critical for the survival of the organism dependent on the pathway involved. However, incapacity in forming a functional Moco has a pleiotropic effect in the different processes involving this cofactor. A detailed overview of Mo metabolism: (a) specific transporters for molybdate, (b) the universal biosynthesis pathway for Moco from GTP, (c) Moco-carrier and Moco-binding proteins for Moco transfer and (d) Mo-enzymes, is analyzed in light of recent findings and three systems are compared, the unicellular microalga Chlamydomonas, the plant Arabidopsis and humans.
Assuntos
Arabidopsis/metabolismo , Chlamydomonas/metabolismo , Molibdênio/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Chlamydomonas/genética , Coenzimas/metabolismo , Humanos , Proteínas de Membrana Transportadoras/classificação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Metaloproteínas/metabolismo , Dados de Sequência Molecular , Cofatores de Molibdênio , Filogenia , Pteridinas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Almost all living organisms need to obtain molybdenum from the external medium to achieve essential processes for life. Activity of important enzymes such as sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase, and nitrate reductase is strictly dependent on the presence of Mo in its active site. Cells take up Mo in the form of the oxianion molybdate, but the molecular nature of the transporters is still not well known in eukaryotes. MOT1 is the first molybdate transporter identified in plant-type eukaryotic organisms, but it is absent in animal genomes. Here we report a molybdate transporter different from the MOT1 family, encoded by the Chlamydomonas reinhardtii gene MoT2, that is also present in animals including humans. The knockdown of CrMoT2 transcription leads to the deficiency of molybdate uptake activity in Chlamydomonas. In addition, heterologous expression in Saccharomyces cerevisiae of MoT2 genes from Chlamydomonas and humans support the functionality of both proteins as molybdate transporters. Characterization of CrMOT2 and HsMOT2 activities showed an apparent Km of about 550 nM that, though higher than the Km reported for MOT1, still corresponds to high affinity systems. CrMoT2 transcription is activated when extracellular molybdate concentration is low but in contrast to MoT1 is not activated by nitrate. Analysis of protein databases revealed the presence of four motifs present in all the proteins with high similarity to MOT2, that label a previously undescribed family of proteins probably related to molybdate transport. Our results open the way toward the understanding of molybdate transport as part of molybdenum homeostasis and Moco biosynthesis in animals.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Chlamydomonas/metabolismo , Molibdênio/metabolismo , Motivos de Aminoácidos , Proteínas de Transporte de Ânions/genética , Chlamydomonas/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Transporte de Íons/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Homeostasis of three elements nickel, molybdenum and chloride is analysed. These micronutrients, at amounts varying in orders of magnitude, fulfil important cell functions. In general terms, cells use similar strategies to ensure that the elements are within physiological ranges avoiding high toxic concentrations. These strategies correspond to specific carriers, channels and pumps, intermediate steps (chelating/sequestration/binding/metabolic conversion/storage), final steps related to specific enzyme functionality and putative sensing proteins. Single cell homeostasis, coordinated with an efficient redistribution by xylem loading, ensures in turn homeostasis at the whole plant level. Recent advances are based on the molecular identification of some key components.