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16 pages, 2039 KiB

Open AccessArticle

Precise Quantification of Molybdate In Vitro by the FRET-Based Nanosensor ‘MolyProbe’

by Kevin D. Oliphant, Marius Karger, Yoichi Nakanishi and Ralf R. Mendel

Molecules 2022, 27(12), 3691; https://doi.org/10.3390/molecules27123691 - 8 Jun 2022

Cited by 4 | Viewed by 1697

Abstract

Molybdenum (Mo) is an essential trace element in all kingdoms of life. Mo is bioavailable as the oxyanion molybdate and gains biological activity in eukaryotes when bound to molybdopterin, forming the molybdenum cofactor. The imbalance of molybdate homeostasis results in growth deficiencies or [...] Read more.

Molybdenum (Mo) is an essential trace element in all kingdoms of life. Mo is bioavailable as the oxyanion molybdate and gains biological activity in eukaryotes when bound to molybdopterin, forming the molybdenum cofactor. The imbalance of molybdate homeostasis results in growth deficiencies or toxic symptoms within plants, fungi and animals. Recently, fluorescence resonance energy transfer (FRET) methods have emerged, monitoring cellular and subcellular molybdate distribution dynamics using a genetically encoded molybdate-specific FRET nanosensor, named MolyProbe. Here, we show that the MolyProbe system is a fast and reliable in vitro assay for quantitative molybdate determination. We added a Strep-TagII affinity tag to the MolyProbe protein for quick and easy purification. This MolyProbe is highly stable, resistant to freezing and can be stored for several weeks at 4 °C. Furthermore, the molybdate sensitivity of the assay peaked at low nM levels. Additionally, The MolyProbe was applied in vitro for quantitative molybdate determination in cell extracts of the plant Arabidopsis thaliana, the fungus Neurospora crassa and the yeast Saccharomyces cerevisiae. Our results show the functionality of the Arabidopsis thaliana molybdate transporter MOT1.1 and indicate that FRET-based molybdate detection is an excellent tool for measuring bioavailable Mo. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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24 pages, 2460 KiB

Open AccessArticle

Physiological Importance of Molybdate Transporter Family 1 in Feeding the Molybdenum Cofactor Biosynthesis Pathway in Arabidopsis thaliana

by Rieke Minner-Meinen, Jan-Niklas Weber, Sarah Kistner, Paul Meyfarth, Merve Saudhof, Lena van den Hout, Jutta Schulze, Ralf-Rainer Mendel, Robert Hänsch and David Kaufholdt

Molecules 2022, 27(10), 3158; https://doi.org/10.3390/molecules27103158 - 15 May 2022

Cited by 9 | Viewed by 2566

Abstract

Molybdate uptake and molybdenum cofactor (Moco) biosynthesis were investigated in detail in the last few decades. The present study critically reviews our present knowledge about eukaryotic molybdate transporters (MOT) and focuses on the model plant Arabidopsis thaliana, complementing it with new experiments, [...] Read more.

Molybdate uptake and molybdenum cofactor (Moco) biosynthesis were investigated in detail in the last few decades. The present study critically reviews our present knowledge about eukaryotic molybdate transporters (MOT) and focuses on the model plant Arabidopsis thaliana, complementing it with new experiments, filling missing gaps, and clarifying contradictory results in the literature. Two molybdate transporters, MOT1.1 and MOT1.2, are known in Arabidopsis, but their importance for sufficient molybdate supply to Moco biosynthesis remains unclear. For a better understanding of their physiological functions in molybdate homeostasis, we studied the impact of mot1.1 and mot1.2 knock-out mutants, including a double knock-out on molybdate uptake and Moco-dependent enzyme activity, MOT localisation, and protein–protein interactions. The outcome illustrates different physiological roles for Moco biosynthesis: MOT1.1 is plasma membrane located and its function lies in the efficient absorption of molybdate from soil and its distribution throughout the plant. However, MOT1.1 is not involved in leaf cell imports of molybdate and has no interaction with proteins of the Moco biosynthesis complex. In contrast, the tonoplast-localised transporter MOT1.2 exports molybdate stored in the vacuole and makes it available for re-localisation during senescence. It also supplies the Moco biosynthesis complex with molybdate by direct interaction with molybdenum insertase Cnx1 for controlled and safe sequestering. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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15 pages, 2548 KiB

Open AccessArticle

The Role of the Nucleotides in the Insertion of the bis-Molybdopterin Guanine Dinucleotide Cofactor into apo-Molybdoenzymes

by Kim Tiedemann, Chantal Iobbi-Nivol and Silke Leimkühler

Molecules 2022, 27(9), 2993; https://doi.org/10.3390/molecules27092993 - 6 May 2022

Cited by 4 | Viewed by 2036

Abstract

The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from [...] Read more.

The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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Review

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20 pages, 2024 KiB

Open AccessReview

Molybdenum Cofactor Deficiency in Humans

by Lena Johannes, Chun-Yu Fu and Günter Schwarz

Molecules 2022, 27(20), 6896; https://doi.org/10.3390/molecules27206896 - 14 Oct 2022

Cited by 16 | Viewed by 3596

Abstract

Molybdenum cofactor (Moco) deficiency (MoCD) is characterized by neonatal-onset myoclonic epileptic encephalopathy and dystonia with cerebral MRI changes similar to hypoxic–ischemic lesions. The molecular cause of the disease is the loss of sulfite oxidase (SOX) activity, one of four Moco-dependent enzymes in men. [...] Read more.

Molybdenum cofactor (Moco) deficiency (MoCD) is characterized by neonatal-onset myoclonic epileptic encephalopathy and dystonia with cerebral MRI changes similar to hypoxic–ischemic lesions. The molecular cause of the disease is the loss of sulfite oxidase (SOX) activity, one of four Moco-dependent enzymes in men. Accumulating toxic sulfite causes a secondary increase of metabolites such as S-sulfocysteine and thiosulfate as well as a decrease in cysteine and its oxidized form, cystine. Moco is synthesized by a three-step biosynthetic pathway that involves the gene products of MOCS1, MOCS2, MOCS3, and GPHN. Depending on which synthetic step is impaired, MoCD is classified as type A, B, or C. This distinction is relevant for patient management because the metabolic block in MoCD type A can be circumvented by administering cyclic pyranopterin monophosphate (cPMP). Substitution therapy with cPMP is highly effective in reducing sulfite toxicity and restoring biochemical homeostasis, while the clinical outcome critically depends on the degree of brain injury prior to the start of treatment. In the absence of a specific treatment for MoCD type B/C and SOX deficiency, we summarize recent progress in our understanding of the underlying metabolic changes in cysteine homeostasis and propose novel therapeutic interventions to circumvent those pathological changes. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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18 pages, 3475 KiB

Open AccessReview

Nitrogenase Fe Protein: A Multi-Tasking Player in Substrate Reduction and Metallocluster Assembly

by Markus W. Ribbe, Kamil Górecki, Mario Grosch, Joseph B. Solomon, Robert Quechol, Yiling A. Liu, Chi Chung Lee and Yilin Hu

Molecules 2022, 27(19), 6743; https://doi.org/10.3390/molecules27196743 - 10 Oct 2022

Cited by 4 | Viewed by 2439

Abstract

The Fe protein of nitrogenase plays multiple roles in substrate reduction and metallocluster assembly. Best known for its function to transfer electrons to its catalytic partner during nitrogenase catalysis, the Fe protein is also a key player in the biosynthesis of the complex [...] Read more.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and metallocluster assembly. Best known for its function to transfer electrons to its catalytic partner during nitrogenase catalysis, the Fe protein is also a key player in the biosynthesis of the complex metalloclusters of nitrogenase. In addition, it can function as a reductase on its own and affect the ambient reduction of CO₂ or CO to hydrocarbons. This review will provide an overview of the properties and functions of the Fe protein, highlighting the relevance of this unique FeS enzyme to areas related to the catalysis, biosynthesis, and applications of the fascinating nitrogenase system. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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7 pages, 2511 KiB

Open AccessReview

Moco Carrier and Binding Proteins

by Tobias Kruse

Molecules 2022, 27(19), 6571; https://doi.org/10.3390/molecules27196571 - 4 Oct 2022

Cited by 5 | Viewed by 1348

Abstract

The molybdenum cofactor (Moco) is the active site prosthetic group found in numerous vitally important enzymes (Mo-enzymes), which predominantly catalyze 2 electron transfer reactions. Moco is synthesized by an evolutionary old and highly conserved multi-step pathway, whereby the metal insertion reaction is the [...] Read more.

The molybdenum cofactor (Moco) is the active site prosthetic group found in numerous vitally important enzymes (Mo-enzymes), which predominantly catalyze 2 electron transfer reactions. Moco is synthesized by an evolutionary old and highly conserved multi-step pathway, whereby the metal insertion reaction is the ultimate reaction step here. Moco and its intermediates are highly sensitive towards oxidative damage and considering this, they are believed to be permanently protein bound during synthesis and also after Moco maturation. In plants, a cellular Moco transfer and storage system was identified, which comprises proteins that are capable of Moco binding and release but do not possess a Moco-dependent enzymatic activity. The first protein described that exhibited these properties was the Moco carrier protein (MCP) from the green alga Chlamydomonas reinhardtii. However, MCPs and similar proteins have meanwhile been described in various plant species. This review will summarize the current knowledge of the cellular Moco distribution system. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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15 pages, 2819 KiB

Open AccessReview

Function of Molybdenum Insertases

by Tobias Kruse

Molecules 2022, 27(17), 5372; https://doi.org/10.3390/molecules27175372 - 23 Aug 2022

Cited by 3 | Viewed by 2115

Abstract

For most organisms molybdenum is essential for life as it is found in the active site of various vitally important molybdenum dependent enzymes (Mo-enzymes). Here, molybdenum is bound to a pterin derivative called molybdopterin (MPT), thus forming the molybdenum cofactor (Moco). Synthesis of [...] Read more.

For most organisms molybdenum is essential for life as it is found in the active site of various vitally important molybdenum dependent enzymes (Mo-enzymes). Here, molybdenum is bound to a pterin derivative called molybdopterin (MPT), thus forming the molybdenum cofactor (Moco). Synthesis of Moco involves the consecutive action of numerous enzymatic reaction steps, whereby molybdenum insertases (Mo-insertases) catalyze the final maturation step, i.e., the metal insertion reaction yielding Moco. This final maturation step is subdivided into two partial reactions, each catalyzed by a distinctive Mo-insertase domain. Initially, MPT is adenylylated by the Mo-insertase G-domain, yielding MPT-AMP which is used as substrate by the E-domain. This domain catalyzes the insertion of molybdate into the MPT dithiolene moiety, leading to the formation of Moco-AMP. Finally, the Moco-AMP phosphoanhydride bond is cleaved by the E-domain to liberate Moco from its synthesizing enzyme. Thus formed, Moco is physiologically active and may be incorporated into the different Mo-enzymes or bind to carrier proteins instead. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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25 pages, 2623 KiB

Open AccessReview

The History of the Molybdenum Cofactor—A Personal View

by Ralf R. Mendel

Molecules 2022, 27(15), 4934; https://doi.org/10.3390/molecules27154934 - 3 Aug 2022

Cited by 15 | Viewed by 4518

Abstract

The transition element molybdenum (Mo) is an essential micronutrient for plants, animals, and microorganisms, where it forms part of the active center of Mo enzymes. To gain biological activity in the cell, Mo has to be complexed by a pterin scaffold to form [...] Read more.

The transition element molybdenum (Mo) is an essential micronutrient for plants, animals, and microorganisms, where it forms part of the active center of Mo enzymes. To gain biological activity in the cell, Mo has to be complexed by a pterin scaffold to form the molybdenum cofactor (Moco). Mo enzymes and Moco are found in all kingdoms of life, where they perform vital transformations in the metabolism of nitrogen, sulfur, and carbon compounds. In this review, I recall the history of Moco in a personal view, starting with the genetics of Moco in the 1960s and 1970s, followed by Moco biochemistry and the description of its chemical structure in the 1980s. When I review the elucidation of Moco biosynthesis in the 1990s and the early 2000s, I do it mainly for eukaryotes, as I worked with plants, human cells, and filamentous fungi. Finally, I briefly touch upon human Moco deficiency and whether there is life without Moco. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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23 pages, 2804 KiB

Open AccessReview

Spectroscopic Studies of Mononuclear Molybdenum Enzyme Centers

by Martin L. Kirk and Russ Hille

Molecules 2022, 27(15), 4802; https://doi.org/10.3390/molecules27154802 - 27 Jul 2022

Cited by 8 | Viewed by 1841

Abstract

A concise review is provided of the contributions that various spectroscopic methods have made to our understanding of the physical and electronic structures of mononuclear molybdenum enzymes. Contributions to our understanding of the structure and function of each of the major families of [...] Read more.

A concise review is provided of the contributions that various spectroscopic methods have made to our understanding of the physical and electronic structures of mononuclear molybdenum enzymes. Contributions to our understanding of the structure and function of each of the major families of these enzymes is considered, providing a perspective on how spectroscopy has impacted the field. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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11 pages, 1005 KiB

Open AccessReview

Beyond Moco Biosynthesis―Moonlighting Roles of MoaE and MOCS2

by Tamaki Suganuma

Molecules 2022, 27(12), 3733; https://doi.org/10.3390/molecules27123733 - 10 Jun 2022

Cited by 1 | Viewed by 2481

Abstract

Molybdenum cofactor (Moco) biosynthesis requires iron, copper, and ATP. The Moco-containing enzyme sulfite oxidase catalyzes terminal oxidation in oxidative cysteine catabolism, and another Moco-containing enzyme, xanthine dehydrogenase, functions in purine catabolism. Thus, molybdenum enzymes participate in metabolic pathways that are essential for cellular [...] Read more.

Molybdenum cofactor (Moco) biosynthesis requires iron, copper, and ATP. The Moco-containing enzyme sulfite oxidase catalyzes terminal oxidation in oxidative cysteine catabolism, and another Moco-containing enzyme, xanthine dehydrogenase, functions in purine catabolism. Thus, molybdenum enzymes participate in metabolic pathways that are essential for cellular detoxication and energy dynamics. Studies of the Moco biosynthetic enzymes MoaE (in the Ada2a-containing (ATAC) histone acetyltransferase complex) and MOCS2 have revealed that Moco biosynthesis and molybdenum enzymes align to regulate signaling and metabolism via control of transcription and translation. Disruption of these functions is involved in the onset of dementia and neurodegenerative disease. This review provides an overview of the roles of MoaE and MOCS2 in normal cellular processes and neurodegenerative disease, as well as directions for future research. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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31 pages, 2601 KiB

Open AccessReview

Inspired by Nature—Functional Analogues of Molybdenum and Tungsten-Dependent Oxidoreductases

by Sebastian Pätsch, Jevy V. Correia, Benedict J. Elvers, Mareile Steuer and Carola Schulzke

Molecules 2022, 27(12), 3695; https://doi.org/10.3390/molecules27123695 - 8 Jun 2022

Cited by 11 | Viewed by 2876

Abstract

Throughout the previous ten years many scientists took inspiration from natural molybdenum and tungsten-dependent oxidoreductases to build functional active site analogues. These studies not only led to an ever more detailed mechanistic understanding of the biological template, but also paved the way to [...] Read more.

Throughout the previous ten years many scientists took inspiration from natural molybdenum and tungsten-dependent oxidoreductases to build functional active site analogues. These studies not only led to an ever more detailed mechanistic understanding of the biological template, but also paved the way to atypical selectivity and activity, such as catalytic hydrogen evolution. This review is aimed at representing the last decade’s progress in the research of and with molybdenum and tungsten functional model compounds. The portrayed systems, organized according to their ability to facilitate typical and artificial enzyme reactions, comprise complexes with non-innocent dithiolene ligands, resembling molybdopterin, as well as entirely non-natural nitrogen, oxygen, and/or sulfur bearing chelating donor ligands. All model compounds receive individual attention, highlighting the specific novelty that each provides for our understanding of the enzymatic mechanisms, such as oxygen atom transfer and proton-coupled electron transfer, or that each presents for exploiting new and useful catalytic capability. Overall, a shift in the application of these model compounds towards uncommon reactions is noted, the latter are comprehensively discussed. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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16 pages, 3238 KiB

Open AccessReview

Synthesis, Redox and Spectroscopic Properties of Pterin of Molybdenum Cofactors

by Kyle J. Colston and Partha Basu

Molecules 2022, 27(10), 3324; https://doi.org/10.3390/molecules27103324 - 22 May 2022

Cited by 2 | Viewed by 2313

Abstract

Pterins are bicyclic heterocycles that are found widely across Nature and are involved in a variety of biological functions. Notably, pterins are found at the core of molybdenum cofactor (Moco) containing enzymes in the molybdopterin (MPT) ligand that coordinates molybdenum and facilitates cofactor [...] Read more.

Pterins are bicyclic heterocycles that are found widely across Nature and are involved in a variety of biological functions. Notably, pterins are found at the core of molybdenum cofactor (Moco) containing enzymes in the molybdopterin (MPT) ligand that coordinates molybdenum and facilitates cofactor activity. Pterins are diverse and can be widely functionalized to tune their properties. Herein, the general methods of synthesis, redox and spectroscopic properties of pterin are discussed to provide more insight into pterin chemistry and their importance to biological systems. Full article

(This article belongs to the Special Issue State-of-the-Art in Molybdenum Cofactor Research)

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