Mcp1 Encodes the Molybdenum Cofactor Carrier Protein in Chlamydomonas reinhardtii and Participates in Protection, Binding, and Storage Functions of the Cofactor

Ataya, Farid S.; Witte, Claus-Peter; Galván, Aurora; Igeño, M. Isabel; Fernández, Emilio

doi:10.1074/jbc.m211320200

Cited by 42 publications

(40 citation statements)

References 34 publications

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“…1A). In summary, MCP binds Moco with high affinity as described earlier (18,20), but this binding is highly specific to Moco and Wco because metal-free MPT is not bound.…”

Section: Selective Binding Of Moco and Wco To Mcp-to Characterizementioning

confidence: 54%

“…Cloning, Expression, and Purification of MCP-CrMCP1 (20) was PCR-cloned into BamHI and HindIII sites of pQE80 yielding pQE80-CrMCP1. To obtain MCP saturated with MPT and/or Moco, we transformed pQE80-CrMCP1 into E. coli strains RK5206 (mogA Ϫ ), TP1000 (mobA Ϫ ), or BL21.…”

Section: Methodsmentioning

confidence: 99%

“…It is unknown whether MCP is also able to donate Moco to molybdenum enzymes other than NR. C. reinhardtii MCP forms a homotetramer in solution and is homologous to bacterial proteins with unknown function (20). The availability of sufficient amounts of Moco is essential for the cell to meet its changing demand of molybdenum enzymes.…”

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confidence: 99%

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Function and Structure of the Molybdenum Cofactor Carrier Protein from Chlamydomonas reinhardtii

Fischer

Llamas

Tejada‐Jiménez

et al. 2006

Journal of Biological Chemistry

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The molybdenum cofactor (Moco) forms the catalytic site in all eukaryotic molybdenum enzymes and is synthesized by a multistep biosynthetic pathway. The mechanism of transfer, storage, and insertion of Moco into the appropriate apo-enzyme is poorly understood. In Chlamydomonas reinhardtii, a Moco carrier protein (MCP) has been identified and characterized recently. Here we show biochemical evidence that MCP binds Moco as well as the tungstate-substituted form of the cofactor (Wco) with high affinity, whereas molybdopterin, the ultimate cofactor precursor, is not bound. This binding selectivity points to a specific metal-mediated interaction with MCP, which protects Moco and Wco from oxidation with t1 ⁄ 2 of 24 and 96 h, respectively. UV-visible spectroscopy showed defined absorption bands at 393, 470, and 570 nm pointing to ene-diothiolate and protein side-chain charge transfer bonds with molybdenum. We have determined the crystal structure of MCP at 1.6 Å resolution using seleno-methionated and native protein. The monomer constitutes a Rossmann fold with two homodimers forming a symmetrical tetramer in solution. Based on conserved surface residues, charge distribution, shape, in silico docking studies, structural comparisons, and identification of an anionbinding site, a prominent surface depression was proposed as a Moco-binding site, which was confirmed by structure-guided mutagenesis coupled to substrate binding studies.Molybdenum is used in the active center of all molybdenum enzymes (1), catalyzing key metabolic reactions in the global sulfur, nitrogen, and carbon cycles in organisms ranging from bacteria to human. With the exception of nitrogenase, in all molybdenum enzymes, molybdenum is found as molybdenum cofactor (Moco) 3 (2) that consists of molybdenum covalently bound to the dithiolate moiety of a tricyclic pterin referred to as molybdopterin or metal-binding pterin (MPT), whose structure is conserved in eukaryotes, eubacteria, and archaea (3). This pterin molecule is also responsible for metal chelation in all tungsten-containing enzymes analyzed so far. Molybdenum enzymes are essential for diverse metabolic processes such as nitrate assimilation in autotrophs and phytohormone synthesis in plants (4) or sulfur detoxification and purine catabolism in mammals (5). Loss of Moco results in the pleiotropic loss of all molybdenum enzymes. Human Moco deficiency is a severe hereditary metabolic disorder (6), and affected patients die in early childhood.In all organisms studied so far, Moco is synthesized by a conserved pathway that can be divided into four major steps (3), according to the biosynthetic intermediates cyclic pyranopterin monophosphate, (formerly described as precursor Z) (7), MPT, and adenylated MPT. In the final and most diverse step of Moco biosynthesis, a single molybdenum atom is ligated to one (in pro-and eukaryotes) or two MPT dithiolates (in prokaryotes).After completion of biosynthesis, mature cofactor has to be inserted into molybdenum enzymes. In prokaryotes, a complex of proteins ...

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“…1A). In summary, MCP binds Moco with high affinity as described earlier (18,20), but this binding is highly specific to Moco and Wco because metal-free MPT is not bound.…”

Section: Selective Binding Of Moco and Wco To Mcp-to Characterizementioning

confidence: 54%

Section: Methodsmentioning

confidence: 99%

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Function and Structure of the Molybdenum Cofactor Carrier Protein from Chlamydomonas reinhardtii

Fischer

Llamas

Tejada‐Jiménez

et al. 2006

Journal of Biological Chemistry

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“…1). Regarding quarternary structure the Arabidopsis homologs, however, differ from Chlamydomonas MCP as they have been crystallized as dimers (19), whereas the latter forms stable homotetramers (9,10). Superposition of Chlamydomonas MCP (Protein Data Bank Code 2IZ5) with the two proteins from Arabidopsis demonstrates a high degree of structural conservation (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Moco containing proteins without a Moco-dependent enzymatic activity, have been suggested to be involved in Moco transfer to apoenzymes (2), but only for the green alga C. reinhardtii are detailed data available. In this alga, the Moco carrier protein MCP binds Moco tightly and stabilizes it against oxidation (9,10). Moreover Moco bound to this protein can be transferred without any denaturing procedure to apoNR from the N. crassa nit-1 mutant.…”

Section: Discussionmentioning

confidence: 99%

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

Kruse

Gehl

Geisler

et al. 2010

Journal of Biological Chemistry

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The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind The molybdenum cofactor (Moco)2 is a prosthetic group highly conserved in all kingdoms of life and consists of a tricyclic pterin, referred to as molybdopterin or metal-binding pterin (MPT) and a molybdenum (Mo) atom covalently bound to the dithiolate moiety of MPT (1). Moco is required for the activity of all Mo-dependent enzymes with the exception of nitrogenase (2). Molybdenum enzymes (Mo-enzymes) are essential for a broad variety of metabolic processes such as nitrate assimilation and phytohormone synthesis in plants (3) and sulfur detoxification and purine catabolism in mammals (4).Synthesis of Moco proceeds in a highly conserved multistep pathway, involving at least six proteins named Cnx in plants (3). Much is known about the final step of Moco biosynthesis where one Mo atom is ligated to the MPT dithiolate function, which is catalyzed by the two-domain protein Cnx1 (5, 6): the C-terminal Cnx1-G domain activates MPT by adenylation, which is handed over to the N-terminal Cnx1-E domain where it is converted to Moco by inserting Mo into MPT under simultaneous cleavage of the pyrophosphate bond.After completion of biosynthesis, Moco has to be allocated and inserted into the apoMo-enzymes. In prokaryotes, a complex of proteins synthesizing the last steps of Moco biosynthesis donates the mature cofactor to apoenzymes assisted by enzyme-specific chaperones (7). In eukaryotes, however, no Mo-enzyme-specific chaperone has been found. As free Moco is extremely sensitive to oxidation it is also assumed that Moco occurs permanently protein-bound in the cell. Therefore, a cellular Moco distribution system should meet two demands: (i) it should bind Moco subsequent to its synthesis, and (ii) it should maintain a directed flow of Moco from the Moco donor Cnx1-E to the Mo-dependent enzymes. This is important to ensure the fast and efficient incorporation of Moco into apoMo-enzymes. In the alga Chlamydomonas reinhardtii a Moco carrier protein (MCP) was identified that was found to bind Moco and protect it against oxidation (8 -10). Without any denaturing procedure, subsequent transfer of Moco from MCP to apo-nitrate reductase (NR) from Neurospora crassa mutant nit-1 was possible (10), thus indicating that MCP-bound Moco was readily transferable. These properties of Chlamydomonas MCP make it a promising candidate for being part of a cellular Moco delivery system. It is, however, unknown whether MCP is also able to donate Moco to Mo-enzymes other than NR.Here we present the cloning and characterization of Mocobinding proteins (MoBP)

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Structure and Function of Xanthine Oxidoreductase

2006

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An overview of the current state of our understanding of the reaction mechanism of the molybdenum‐containing enzyme xanthine oxidoreductase is presented, with an emphasis on work done in the past five years. Recent studies of the biosynthesis of the pterin cofactor bound to the metal in the active site are also reviewed, as is crystallographic work that has clarified the coordination geometry of the molybdenum center. This structural work provides the context in which to understand recent mechanistic studies of the enzyme, in particular those aimed at elucidating the role of specific amino acid residues in the active site of the enzyme.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

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Mcp1 Encodes the Molybdenum Cofactor Carrier Protein in Chlamydomonas reinhardtii and Participates in Protection, Binding, and Storage Functions of the Cofactor

Cited by 42 publications

References 34 publications

Function and Structure of the Molybdenum Cofactor Carrier Protein from Chlamydomonas reinhardtii

Function and Structure of the Molybdenum Cofactor Carrier Protein from Chlamydomonas reinhardtii

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

Structure and Function of Xanthine Oxidoreductase

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