Moco Deficiency Research Articles

Convergent evolution links molybdenum insertase domains with organism-specific sequences

In all domains of life, the biosynthesis of the pterin-based Molybdenum cofactor (Moco) is crucial. Molybdenum (Mo) becomes biologically active by integrating into a unique pyranopterin scaffold, forming Moco. The final two steps of Moco biosynthesis are catalyzed by the two-domain enzyme Mo insertase, linked by gene fusion in higher organisms. Despite well-understood Moco biosynthesis, the evolutionary significance of Mo insertase fusion remains unclear. Here, we present findings from Neurospora crassa that shed light on the critical role of Mo insertase fusion in eukaryotes. Substituting the linkage region with sequences from other species resulted in Moco deficiency, and separate expression of domains, as seen in lower organisms, failed to rescue deficient strains. Stepwise truncation and structural modeling revealed a crucial 20-amino acid sequence within the linkage region essential for fungal growth. Our findings highlight the evolutionary importance of gene fusion and specific sequence composition in eukaryotic Mo insertases.

Obtaining the necessary molybdenum cofactor for sulfite oxidase activity in the nematode Caenorhabditis elegans surprisingly involves a dietary source

Molybdenum cofactor (Moco) is a prosthetic group necessary for the activity of four unique enzymes, including the essential sulfite oxidase (SUOX-1). Moco is required for life; humans with inactivating mutations in the genes encoding Moco-biosynthetic enzymes display Moco deficiency, a rare and lethal inborn error of metabolism. Despite its importance to human health, little is known about how Moco moves among and between cells, tissues, and organisms. The prevailing view is that cells that require Moco must synthesize Moco de novo. Although, the nematode Caenorhabditis elegans appears to be an exception to this rule and has emerged as a valuable system for understanding fundamental Moco biology. C.elegans has the seemingly unique capacity to both synthesize its own Moco as well as acquire Moco from its microbial diet. However, the relative contribution of Moco from the diet orendogenous synthesis has not been rigorously evaluated or quantified biochemically. We genetically removed dietary or endogenous Moco sources in C.elegans and biochemically determined their impact on animal Moco content and SUOX-1 activity. We demonstrate that dietary Moco deficiency dramatically reduces both animal Moco content and SUOX-1 activity. Furthermore, these biochemical deficiencies have physiological consequences; we show that dietary Moco deficiency alone causes sensitivity to sulfite, the toxic substrate of SUOX-1. Altogether, this work establishes the biochemical consequences of depleting dietary Moco or endogenous Moco synthesis in C.elegans and quantifies the surprising contribution of the diet to maintaining Moco homeostasis in C.elegans.

Molybdenum Cofactor Deficiency in Humans.

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.

The History of the Molybdenum Cofactor-A Personal View.

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.

Moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans.

Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency, a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/moc-6, the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/moc-6 and with these alleles genetically demonstrate that K10D2.7/moc-6 is necessary for endogenous Moco synthesis in C. elegans.

Resolving the Multidecade-Long Mystery in MoaA Radical SAM Enzyme Reveals New Opportunities to Tackle Human Health Problems.

MoaA is one of the most conserved radical S-adenosyl-l-methionine (SAM) enzymes, and is found in most organisms in all three kingdoms of life. MoaA contributes to the biosynthesis of molybdenum cofactor (Moco), a redox enzyme cofactor used in various enzymes such as purine and sulfur catabolism in humans and anaerobic respiration in bacteria. Unlike many other cofactors, in most organisms, Moco cannot be taken up as a nutrient and requires de novo biosynthesis. Consequently, Moco biosynthesis has been linked to several human health problems, such as human Moco deficiency disease and bacterial infections. Despite the medical and biological significance, the biosynthetic mechanism of Moco’s characteristic pyranopterin structure remained elusive for more than two decades. This transformation requires the actions of the MoaA radical SAM enzyme and another protein, MoaC. Recently, MoaA and MoaC functions were elucidated as a radical SAM GTP 3′,8-cyclase and cyclic pyranopterin monophosphate (cPMP) synthase, respectively. This finding resolved the key mystery in the field and revealed new opportunities in studying the enzymology and chemical biology of MoaA and MoaC to elucidate novel mechanisms in enzyme catalysis or to address unsolved questions in Moco-related human health problems. Here, we summarize the recent progress in the functional and mechanistic studies of MoaA and MoaC and discuss the field’s future directions.

Learning from the worm: the effectiveness of protein-bound Moco to treat Moco deficiency.

Molybdenum cofactor (Moco) is synthesized endogenously in humans and is essential for human development. Supplementation of Moco or its precursors has been explored as a therapy to treat Moco-deficient patients but with significant limitations. By using the nematode C. elegans as a model, Warnhoff and colleagues (pp. 212-217) describe the beneficial impact of protein-bound Moco supplementation to treat Moco deficiency. If such an effect is conserved, this advance from basic research in worms may have significant clinical implications as a novel therapy for molybdenum cofactor deficiency.

Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.

The molybdenum cofactor (Moco) is a 520-Da prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy; however, the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a Celegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Molybdenum cofactor biology, evolution and deficiency

The molybdenum cofactor (Moco) represents an ancient metal‑sulfur cofactor, which participates as catalyst in carbon, nitrogen and sulfur cycles, both on individual and global scale. Given the diversity of biological processes dependent on Moco and their evolutionary age, Moco is traced back to the last universal common ancestor (LUCA), while Moco biosynthetic genes underwent significant changes through evolution and acquired additional functions. In this review, focused on eukaryotic Moco biology, we elucidate the benefits of gene fusions on Moco biosynthesis and beyond. While originally the gene fusions were driven by biosynthetic advantages such as coordinated expression of functionally related proteins and product/substrate channeling, they also served as origin for the development of novel functions. Today, Moco biosynthetic genes are involved in a multitude of cellular processes and loss of the according gene products result in severe disorders, both related to Moco biosynthesis and secondary enzyme functions.

ETHE1 and MOCS1 deficiencies: Disruption of mitochondrial bioenergetics, dynamics, redox homeostasis and endoplasmic reticulum-mitochondria crosstalk in patient fibroblasts

Ethylmalonic encephalopathy protein 1 (ETHE1) and molybdenum cofactor (MoCo) deficiencies are hereditary disorders that affect the catabolism of sulfur-containing amino acids. ETHE1 deficiency is caused by mutations in the ETHE1 gene, while MoCo deficiency is due to mutations in one of three genes involved in MoCo biosynthesis (MOCS1, MOCS2 and GPHN). Patients with both disorders exhibit abnormalities of the mitochondrial respiratory chain, among other biochemical findings. However, the pathophysiology of the defects has not been elucidated. To characterize cellular derangements, mitochondrial bioenergetics, dynamics, endoplasmic reticulum (ER)-mitochondria communication, superoxide production and apoptosis were evaluated in fibroblasts from four patients with ETHE1 deficiency and one with MOCS1 deficiency. The effect of JP4-039, a promising mitochondrial-targeted antioxidant, was also tested on cells. Our data show that mitochondrial respiration was decreased in all patient cell lines. ATP depletion and increased mitochondrial mass was identified in the same cells, while variable alterations in mitochondrial fusion and fission were seen. High superoxide levels were found in all cells and were decreased by treatment with JP4-039, while the respiratory chain activity was increased by this antioxidant in cells in which it was impaired. The content of VDAC1 and IP3R, proteins involved in ER-mitochondria communication, was decreased, while DDIT3, a marker of ER stress, and apoptosis were increased in all cell lines. These data demonstrate that previously unrecognized broad disturbances of cellular function are involved in the pathophysiology of ETHE1 and MOCS1 deficiencies, and that reduction of mitochondrial superoxide by JP4-039 is a promising strategy for adjuvant therapy of these disorders.

Analysis of the Cellular Roles of MOCS3 Identifies a MOCS3-Independent Localization of NFS1 at the Tips of the Centrosome.

The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the l-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm5s2U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm5s2U thio-modified tRNAs were not detectable. Because the l-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.

Mouse model for molybdenum cofactor deficiency type B recapitulates the phenotype observed in molybdenum cofactor deficient patients.

Molybdenum cofactor (MoCo) deficiency is a rare, autosomal-recessive disorder, mainly caused by mutations in MOCS1 (MoCo deficiency type A) or MOCS2 (MoCo deficiency type B) genes; the absence of active MoCo results in a deficiency in all MoCo-dependent enzymes. Patients with MoCo deficiency present with neonatal seizures, feeding difficulties, severe developmental delay, brain atrophy and early childhood death. Although substitution therapy with cyclic pyranopterin monophosphate (cPMP) has been successfully used in both Mocs1 knockout mice and in patients with MoCo deficiency type A, there is currently no Mocs2 knockout mouse and no curative therapy for patients with MoCo deficiency type B. Therefore, we generated and characterized a Mocs2-null mouse model of MoCo deficiency type B. Expression analyses of Mocs2 revealed a ubiquitous expression pattern; however, at the cellular level, specific cells show prominent Mocs2 expression, e.g., neuronal cells in cortex, hippocampus and brainstem. Phenotypic analyses demonstrated that Mocs2 knockout mice failed to thrive and died within 11days after birth. None of the tested MoCo-dependent enzymes were active in Mocs2-deficient mice, leading to elevated concentrations of purines, such as hypoxanthine and xanthine, and non-detectable levels of uric acid in the serum and urine. Moreover, elevated concentrations of S-sulfocysteine were measured in the serum and urine. Increased levels of xanthine resulted in bladder and kidney stone formation, whereas increased concentrations of toxic sulfite triggered neuronal apoptosis. In conclusion, Mocs2-deficient mice recapitulate the severe phenotype observed in humans and can now serve as a model for preclinical therapeutic approaches for MoCo deficiency type B.

Structural Framework for Metal Incorporation during Molybdenum Cofactor Biosynthesis

The molybdenum cofactor (Moco) is essential forthecatalytic activity of all molybdenum-containing enzymes with the exception of nitrogenase. Moco biosynthesis follows an evolutionarily highly conserved pathway and genetic deficiencies in the corresponding human enzymes result in Moco deficiency, which manifests itself in severe neurological symptoms and death in childhood. In humans the final steps of Moco biosynthesis are catalyzed by gephyrin, specifically the penultimate adenylation of molybdopterin (MPT) by its N-terminal G domain (GephG) and the final metal incorporation by its C-terminal E domain (GephE). To better understand the poorly defined molecular framework of this final step, we determined high-resolution crystal structures of GephE in the apo state and in complex with ADP, AMP, and molybdate. Our data provide novel insights into the catalytic steps leading to final Moco maturation, namely deadenylation as well as molybdate binding and insertion.

C-Terminal glycine-gated radical initiation by GTP 3',8-cyclase in the molybdenum cofactor biosynthesis.

The molybdenum cofactor (Moco) is an essential redox cofactor found in all kingdoms of life. Genetic mutations in the human Moco biosynthetic enzymes lead to a fatal metabolic disorder, Moco deficiency (MoCD). Greater than 50% of all human MoCD patients have mutations in MOCS1A, a radical S-adenosyl-l-methionine (SAM) enzyme involved in the conversion of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin monophosphate. In MOCS1A, one of the frequently affected locations is the GG motif constituted of two consecutive Gly at the C-terminus. The GG motif is conserved among all MOCS1A homologues, but its role in catalysis or the mechanism by which its mutation causes MoCD was unknown. Here, we report the functional characterization of the GG motif using MoaA, a bacterial homologue of MOCS1A, as a model. Our study elucidated that the GG motif is essential for the activity of MoaA to produce 3',8-cH2GTP from GTP (GTP 3',8-cyclase), and that synthetic peptides corresponding to the C-terminal region of wt-MoaA rescue the GTP 3',8-cyclase activity of the GG-motif mutants. Further biochemical characterization suggested that the C-terminal tail containing the GG motif interacts with the SAM-binding pocket of MoaA, and is essential for the binding of SAM and subsequent radical initiation. In sum, these observations suggest that the C-terminal tail of MoaA provides an essential mechanism to trigger the free radical reaction, impairment of which results in the complete loss of catalytic function of the enzyme, and causes MoCD.

Characterization of Chlamydomonas 102 and 104 Mutants Reveals Intermolecular Complementation in the Molybdenum Cofactor Protein CNX1E

Chlamydomonas reinhardtii is a eukaryotic, unicellular, biflagellate green alga. In Chlamydomonas nitrate assimilation depends on the activity of the enzyme nitrate reductase that requires the molybdenum cofactor (Moco). We have characterized the Chlamydomonas strains 102 (nit5nit6), 104 (nit4nit5) and 106 (nit6). These mutations lead to the exchanges V171A (nit4) and G183D (nit6) in the CNX1E protein of Moco biosynthesis that result in Moco deficiency and hence inability to grow on nitrate. CNX1E inserts molybdenum into molybdopterin (MPT) to yield Moco through the intermediate MPT-AMP. The CNX1E variant G183D is unable to bind MPT-AMP. However, the CNX1E variant V171A is able to bind MPT-AMP but is not able to hydrolyze it and insert the molybdenum to obtain Moco. We show that intermolecular complementation between the CNX1E variants V171A and G183D both in vivo and in vitro restored its activity. The molecular characterization of strain 106 led us to discover the presence of wild type and nit6 CNX1E alleles in this strain (nit6/+). Then, we found out that 106 is indeed a diploid strain. It is proposed that 102, the strain from which 106 derived, may contain a genetic alteration that produces an aberrant meiotic division of the mature zygotes.

Molybdenum cofactor deficiency: Mutations in GPHN, MOCS1, and MOCS2

All molybdenum-containing enzymes other than the bacterial nitrogenase share an identical molybdenum cofactor (MoCo), which is synthesized via a conserved pathway in all organisms and therefore also is called "universal molybdenum cofactor." In humans, four molybdoenzymes are known: aldehyde oxidase, mitochondrial amidoxime reducing component (mARC), xanthine oxidoreductase, and sulfite oxidase. Mutations in the genes encoding the biosynthetic MoCo pathway enzymes abrogate the activities of all molybdoenzymes and result in the "combined" form of MoCo deficiency, which is clinically very similar to isolated sulfite oxidase deficiency, caused by mutations in the gene for the corresponding apoenzyme. Both deficiencies are inherited as an autosomal-recessive disease and result in progressive neurological damage and early childhood death in most cases. The majority of mutations leading to MoCo deficiency have been identified in the genes MOCS1 (type A deficiency), MOCS2 (type B deficiency), with one reported in GPHN. For type A deficiency an effective substitution therapy has been described recently.

Chronological changes of the amplitude‐integrated EEG in a neonate with molybdenum cofactor deficiency

Molybdenum cofactor (Moco) deficiency is a rare neurometabolic disorder, characterized by neurological impairment and refractive seizures, due to toxic accumulation of sulfite in the brain. Earlier it was suggested that in Moco-deficient humans maternal clearance of neurotoxic metabolites prevents prenatal brain damage. However, limited data are available about the time profile in which neurophysiologic deterioration occurs after birth. The amplitude-integrated electroencephalography (aEEG) is a bedside method in neonates to monitor cerebral recovery after hypoxic-ischemic insults, detect epileptic activity, and evaluate antiepileptic drug treatment. We describe a chronological series of changes in aEEG tracings in a neonate with Moco deficiency. He presented with myoclonic spasms and hypertonicity a few hours after birth, however, the aEEG pattern was still normal. Within 2 days, the aEEG rapidly changed into a burst suppression pattern with repetitive seizures. After antiepileptic treatment, the aEEG remained abnormal. In this patient, the normal aEEG pattern at birth may have been due to maternal clearance of sulfite in utero. After birth, accumulation of sulfite causes progressive brain damage, reflected by the progressive depression of the aEEG tracings. This is in agreement with the results from a Moco-deficient mouse model, suggesting that maternal sulfite clearance suppresses prenatal brain damage. To our knowledge, this is the first case report describing the chronological changes in the aEEG pattern in a Moco-deficient patient. Insight into the time profile in which neurologic deterioration in Moco-deficient humans occurs is essential, especially when potential treatment strategies are being evaluated.

Maternal uniparental isodisomy is responsible for serious molybdenum cofactor deficiency

Molybdenum cofactor (MoCo) deficiency is a rare autosomal recessive inherited metabolic disorder resulting in the combined deficiency of aldehyde oxidase, xanthine dehydrogenase, and sulfite oxidase. We report a male infant with MoCo deficiency whose clinical findings consisted of microcephaly, intractable seizures soon after birth, feeding difficulties, and developmental delay. Sequencing of MOCS1, MOCS2, and GEPH genes, and single nucleotide polymorphism genotyping array analysis showed, to our knowledge, unusual inheritance of MoCo deficiency/maternal uniparental isodisomy for the first time in the literature. At 10 months of age, he now has microcephaly and developmental delay, and his seizures are controlled with phenobarbital, clonozepam, and vigabatrin therapy.

Long-Term Rescue of a Lethal Inherited Disease by Adeno-Associated Virus–Mediated Gene Transfer in a Mouse Model of Molybdenum-Cofactor Deficiency

Molybdenum cofactor (MoCo) deficiency is a progressive neurological disorder that inevitably leads to early childhood death because of the lack of any effective therapy. In a mouse model of MoCo deficiency type A, the most frequent form of this autosomal recessively inherited disease, the affected animals show the biochemical characteristics of sulphite and xanthine intoxication and do not survive >2 wk after birth. We have constructed a recombinant-expression cassette for the gene MOCS1, which, via alternative splicing, facilitates the expression of the proteins MOCS1A and MOCS1B, both of which are necessary for the formation of a first intermediate, cyclic pyranopterin monophosphate (cPMP), within the biosynthetic pathway leading to active MoCo. A recombinant adeno-associated virus (AAV) vector was used to express the artificial MOCS1 minigene, in an attempt to cure the lethal MOCS1-deficient phenotype. The vector was used to transduce Mocs1-deficient mice at both 1 and 4 d after birth or, after a pretreatment with purified cPMP, at 40 d after birth. We report here that all Mocs1-deficient animals injected with a control AAV-enhanced green fluorescent protein vector died approximately 8 d after birth or after withdrawal of cPMP supplementation, whereas AAV-MOCS1-transduced animals show significantly increased longevity. A single intrahepatic injection of AAV-MOCS1 resulted in fertile adult animals without any pathological phenotypes.

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

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 t((1/2)) 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 Angstrom 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.