Direct transfer of molybdopterin cofactor to aponitrate reductase from a carrier protein in <i>Chlamydomonas reinhardtii</i>

Aguilar, Miguel; Kalakoutskii, Kyrill; Cárdenas, Jacobo; Fernández, Emilio

doi:10.1016/0014-5793(92)80758-9

Cited by 35 publications

(42 citation statements)

References 15 publications

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“…In light of the different concentrations used in both systems (10 M Moco versus 4 nM MCP), the transfer of Moco to apo-NR can be considered as fast and efficient. When also taking into account that Moco binds with high affinity to MCP (due to its efficient co-purification), the observed transfer rates would nevertheless argue for a direct insertion of Moco from MCP into apo-NR via protein-protein interactions as indicated by previous studies (13) This conclusion is further supported by our inhibition studies with tungstate. Due to the high affinity binding of Moco to MCP, one can conclude that binding and storage of Moco is one of the crucial functions of MCP in Chlamydomonas metabolism.…”

Section: Discussionsupporting

confidence: 85%

“…In eukaryotes, no molybdenum enzyme-specific chaperone has been found until now, and direct transfer of Moco is possible at least in vitro (10). However, in several organisms such as Escherichia coli (11), plant seeds (12) and the green alga Chlamydomonas reinhardtii (13), Moco-binding proteins were found. Due to the two known classes of eukaryotic molybdenum enzymes (1), differences in the insertion of Moco might also be considered.…”

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confidence: 99%

“…Among eukaryotes, the activity of a Moco carrier protein (MCP) was described at first in the green alga C. reinhardtii (13). Later, by following this activity, a 16-kDa protein was purified and shown to bind and protect Moco against oxidation (18).…”

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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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Section: Discussionsupporting

confidence: 85%

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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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“…In E. coli, Moco has been shown to be protected by tight binding to a 40-kDa Moco carrier protein (41). A similar situation has been encountered in Chlamydomonas reinhardtii with a 50-kDa carrier protein (44,45 The bacterial two-hybrid approach described here constitutes an essential tool to address such a fundamental question.…”

Section: Discussionmentioning

confidence: 72%

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

Magalon

Frixon

Pommier

et al. 2002

Journal of Biological Chemistry

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The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin. Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli. Numerous interactions among these proteins have been identified. Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA. Probing the effects of various mo. mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions. These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins.

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“…In a previous study (13), we purified a Moco-binding protein from Chlamydomonas reinhardtii. It was named molybdenum cofactor carrier protein (MocoCP) because of its ability to transfer Moco directly to aponitrate reductase (14). This 64-kDa MocoCP with four identical subunits of 16.5 kDa protected Moco against inactivation under aerobic conditions and basic pH levels.…”

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confidence: 99%

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

Ataya

Witte²,

Galván

et al. 2003

Journal of Biological Chemistry

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Direct transfer of molybdopterin cofactor to aponitrate reductase from a carrier protein in Chlamydomonas reinhardtii

Cited by 35 publications

References 15 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

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

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

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