Search

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt

An efficient synthesis of the molybdopterin/molybdenum cofactor (Moco) oxidation product dephospho Form A is described that assembles the pteridinone system starting from an iodinated aminopyrazine. The sodium salt of dephospho Form A could be purified by precipitation from methanol, which paved the way to the title compound in the 100 mg range. By HPLC, the synthetic material was compared with a sample isolated from a recombinant Moco containing protein. Analysis of dephospho Form A is the only method that allows the quantification of the Moco content of crude cell extracts and recombinant protein preparations.

Background Molybdenum cofactor deficiency (MoCD) is a rare autosomal‐recessive disorder that results in the combined deficiency of molybdenum‐dependent enzymes. Four different genes are involved in Molybdenum cofactor biosynthesis: MOCS1, MOCS2, MOCS3, and GEPH. The classical form manifests in the neonatal period with severe encephalopathy, including intractable seizures, MRI changes that resemble hypoxic‐ischemic injury, microcephaly, and early death. To date, an atypical phenotype with late‐onset has been reported in the literature in 13 patients. Methods We describe a late‐onset and a relatively mild phenotype in a patient with MOCS2 homozygous mutation. Results Pyramidal and extrapyramidal signs are recognized in those patients, often exacerbated by intercurrent illness. Expressive language is usually compromised. Neurological deterioration is possible even in adulthood, probably due to accumulation of sulfite with time. Conclusion Sulfite inhibition of mitochondrial metabolism could be responsible for the ischemic lesions described in patients with MoCD or alternatively could predispose the brain to suffer an ischemic damage through the action of other insults, for instance intercurrent illness. It is possible that sulfite accumulation together with other external triggers, can lead to neurological deterioration even in adulthood. The role of other factors involved in clinical expression should be investigated to establish the reason for phenotypic variability in patients with the same mutation.

Molybdoenzymes contain a molybdenum cofactor in their active site to catalyze various redox reactions in all domains of life. To decipher crucial steps during their biogenesis, the TorA molybdoenzyme of Escherichia coli had played a major role to understand molybdoenzyme maturation process driven by specific chaperones. TorD, the specific chaperone of TorA, is also involved in TorA protection. Here, we show that immature TorA (apoTorA) is degraded in vivo and in vitro by the Lon protease. Lon interacts with apoTorA but not with holoTorA. Lon and TorD compete for apoTorA binding but TorD binding protects apoTorA against degradation. Lon is the first protease shown to eliminate an immature or misfolded molybdoenzyme probably by targeting its inactive catalytic site.

Molybdenum (Mo) is an essential micronutrient for almost all organisms. In eukaryotes, it forms a part of the molybdenum cofactor (Moco), which is required for numerous enzymes involved in carbon, nitrogen and sulfur metabolism. Mo is taken up by cells in the form of molybdate and recently molybdate transporters have been identified in Arabidopsis thaliana and Chlamydomonas reinhardtii. Here, we report the characterization of a novel mutant (DB6) of C. reinhardtii generated by random insertional mutagenesis that is unable to assimilate nitrate as a nitrogen source because it lacks functional nitrate reductase (NR). Besides lacking NR, DB6 also lacks xanthine dehydrogenase activity; a common requirement of both enzymes is Moco. DB6 displays a 'molybdate-repairable' phenotype--growth on nitrate is partially restored by supplementing media with high levels of molybdate. This phenotype is typically associated with mutants defective in either molybdate transport or insertion of Mo into the pterin precursor of Moco. Mo content was found to be significantly lower in DB6 than in the wild-type strain, AOXR1, which suggests that DB6 is defective in Mo uptake. Genetic complementation with a variety of candidate genes that include the known molybdate transporter MOT1 and DNA that spans the site of mutation was unable to recover the wild-type phenotype. Taken together, our results indicate that DB6 is a novel molybdate transport-deficient mutant.

We have identified a highly conserved RNA motif located upstream of genes encoding molybdate transporters, molybdenum cofactor (Moco) biosynthesis enzymes, and proteins that utilize Moco as a coenzyme. Bioinformatics searches have identified 176 representatives in gamma-Proteobacteria, delta-Proteobacteria, Clostridia, Actinobacteria, Deinococcus-Thermus species and DNAs from environmental samples. Using genetic assays, we demonstrate that a Moco RNA in Escherichia coli associated with the Moco biosynthetic operon controls gene expression in response to Moco production. In addition, we provide evidence indicating that this conserved RNA discriminates against closely related analogues of Moco. These results, together with extensive phylogenetic conservation and typical gene control structures near some examples, indicate that representatives of this structured RNA represent a novel class of riboswitches that sense Moco. Furthermore, we identify variants of this RNA that are likely to be triggered by the related tungsten cofactor (Tuco), which carries tungsten in place of molybdenum as the metal constituent.

Pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici, a molybdopterin-containing enzyme, catalyzes a key reaction in the anaerobic degradation of aromatic compounds. In vitro, the enzymatic reaction requires 1,2,3,5-tetrahydroxybenzene as a cocatalyst and the transhydroxylation occurs without exchange with hydroxy groups from water. To test our previous proposal that the transfer of the hydroxy group occurs via 2,4,6,3',4',5'-hexahydroxydiphenyl ether as an intermediate, we synthesized this compound and investigated its properties. We also describe the synthesis and characterization of 3,4,5,3',4',5'-hexahydroxydiphenyl ether. Both compounds could substitute for the cocatalyst in vitro. This indicates that the diphenyl ethers can intrude into the active site and initiate the catalytic cycle. Recently, the X-ray crystal structure of the transhydroxylase (TH) was published [16] and it supports the proposed mechanism of hydroxy-group transfer.

†Summary Abscisic acid (ABA), auxin and nitrate are important signaling molecules that affect plant growth responses to the environment. The synthesis or metabolism of these compounds depends on the molybdenum cofactor (MoCo). We show that maize (Zea mays) viviparous10 (vp10) mutants have strong precocious germination and seedling lethal phenotypes that cannot be rescued with tissue culture. We devised a novel PCR-based method to clone a transposon-tagged allele ofvp10, and show that Vp10 encodes the ortholog of Cnx1, which catalyzes the final common step of MoCo synthesis. The seedling phenotype of vp10 mutants is consistent with disruptions in ABA and auxin biosynthesis, as well as a disruption in nitrate metabolism. Levels of ABA and auxin are reduced in vp10 mutants, and vp10 seedlings lack MoCo-dependent enzyme activities that are repairable with exogenous molybdenum. vp10 and an Arabidopsis cnx1 mutant, chlorate6 (chl6), have similar defects in aldehyde oxidase (AO) enzyme activity, which is required for ABA synthesis. Surprisingly, chl6 mutants do not show defects in abiotic stress responses. These observations confirm an orthologous function for Cnx1 and Vp10, as well as defining a characteristic viviparous phenotype to identify other maize cnx mutants. Finally, the vp10 mutant phenotype suggests that cnx mutants can have auxin- as well as ABAbiosynthesis defects, while the chl6 mutant phenotype suggests that low levels of AO activity are sufficient for normal abiotic stress responses.

Molybdenum cofactor and isolated sulphite oxidase deficiencies are two related rare autosomal recessive diseases characterized by severe neurological abnormalities, dislocated lens and mental retardation. Determination of three biochemical markers S-sulphocysteine (SSC), xanthine (XAN) and hypoxanthine (HXAN) in urine is essential for a definitive diagnosis and identification of the exact defect. We developed a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of SSC, XAN and HXAN in urine. The analysis was carried out in the negative-ion selected-reaction monitoring mode. The turnaround time for the assay was 7 min. Linear calibration curves for the three biomarkers were obtained in the range of 12-480 micromol/L. The intra- and inter-day assay variations were

Maria Solà et al., Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR β-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the β-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity., This work was supported by EMBL (WW), Deutsche Forschungsgemeinschaft (SFB 628) and ‘Fonds der Chemischen Industrie’ (HB). VB was supported by a predoctoral fellowship from the ‘Louis-Jeantet Fondation de Medicine’ and MS and GAO’S were both supported by a Marie Curie fellowship from the European Union

The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand. In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position. Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre. The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor. Key similarities were found with NifS, the enzyme involved in the generation of the iron-sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulphur ligand. We have identified putative homologues of these genes in a variety of organisms, including humans. The human homologue is located in chromosome 18.q12.

The molybdenum-containing iron-sulfur flavoprotein CO dehydrogenase is expressed in a catalytically fully competent form during heterotrophic growth of the aerobic bacterium Hydrogenophaga pseudoflava with pyruvate plus CO. We have adopted these conditions for studying the effect of molybdate (Mo) and tungstate (W) on the biosynthesis of CO dehydrogenase and its molybdopterin (MPT) cytosine-dinucleotide-(MCD)-type molybdenum cofactor. W was taken up by the Mo transport system and, therefore, interfered with Mo transport in an antagonistic way. Depletion of Mo from the growth medium as well as inclusion of excess W both resulted in the absence of intracellular Mo and led to the biosynthesis of CO dehydrogenase species of proper L2M2S2 subunit structure that carried the two 2Fe : 2S type-I and type-II centers and two FAD molecules. EPR, ultraviolet/visible and CD spectroscopies established the full functionality of the cofactors. Due to the absence of the Mo-MCD cofactor, the enzyme species were catalytically inactive. Unexpectedly, the following cytidine nucleotides were present in inactive CO dehydrogenase : CDP, dCDP, CMP, dCMP, CTP or dCTP. The sum of cytidine nucleotides was two/mol enzyme. The binding specificities of inactive CO dehydrogenase for cytidine nucleotides (oxy > deoxy; diphosphate > monophosphate > triphosphate), and the absence of MPT suggest that, in active CO dehydrogenase, the cytidine diphosphate moiety of Mo-MCD provides the strongest interactions with the protein and determines the specificity for the type of nucleotide. In H. pseudoflava, the biosynthesis of MPT (identified as form A) was independent of Mo. Mo was, however, strictly required for the conversion of MPT to MCD (identified as form-A−CMP) as well as the insertion of Mo-MCD into CO dehydrogenase. These data support a model for the involvement of Mo in the biosynthesis of the Mo-MCD cofactor and of fully functional CO dehydrogenase in which the synthesis and insertion of Mo-MCD require Mo, and protein synthesis including integration of the FeS-centers and FAD are independent of Mo.

In Chlamydomonas reinhardtii, molybdopterin cofactor (MoCo) able to reconstitute active nitrate reductase (NR) with apoenzyme from the Neurospora crassa mutant nit-1 was found mostly bound to a carrier protein (CP). This protein is scarce in the algal free extracts and has been purified 520-fold. MoCoCP is a protein of 64 kDa with subunits of 16.5 kDa and an isoelectric point of 4.5. In contrast to free MoCo, MoCo bound to CP was remarkably protected against inactivation under both aerobic conditions and basic pH. MocoCP transferred active MoCo to apoNR in vitro without addition of molybdate, though reconstituted activity was 20% higher in the presence of molybdate. Incubation with tungstate specifically inhibited MoCoCP activity but had no effect on the activity of free MoCo released from milk xanthine oxidase. MoCoCP did not charge molybdate unless in the presence of N. crassa extracts. Our data support that MoCoCP stabilizes MoCo in an active form charged with molybdate to provide MoCo to apomolybdoenzymes.

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro. NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.

A gene cluster consisting of homologs to Escherichia coli moaA, moeA, moaC and moaE, which encode enzymes involved in the biosynthesis of molybdopterin cofactor (MoCo), and to modA, modB and modC, which encode a high-affinity molybdate transporter, were identified on pAO1 of Arthrobacter nicotinovorans near genes of molybdopterin-dependent enzymes involved in nicotine degradation. This gene arrangement suggests a coordinated expression of the MoCo-dependent and the MoCo-biosynthesis genes and shows that catabolic plasmids may carry the transport and biosynthetic machinery for the synthesis of the cofactors needed for the functioning of the enzymes they encode. pAO1 MoeA functionally complemented E. coli moeA mutants. The overexpressed and purified protein, of molecular mass 44,500 Da, associated into high-molecular-mass complexes and spontaneously formed gels at concentrations above 1 mg/ml. Transmission electron microscopy and atomic force microscopy revealed that MoeA forms fibrilar structures. In the presence of Mg2+ MoeA exhibited ATPase activity (0.020 pmol ATP x pmol protein(-1) x min(-1)). ATP, ADP or AMP induced the disassembly of the MoeA fibers into aggregates. pAO1 MoeA shows 39% identity to the C-terminal domain of the rat neuroprotein gephyrin. Like gephyrin it binds to neurotubulin, but binds with preference to tubulin dimers.

Analysis of dimethylsulfoxide reductase from Rhodobacter capsulatus showed that it contained 1 mol Mo and 2 mol GMP. This indicates that the molybdenum cofactor in dimethylsulfoxide reductase is bis(molybdopterin guanine dinucleotide) molybdenum. The absorption spectrum of the molybdopterin guanine dinucleotide released from dimethylsulfoxide reductase after denaturation of the holoenzyme was compared with those of pterin standards of known redox state. The spectra were most similar to pterin standards in the dihydro state and oxidised state. The reduction of 2,6-dichloroindophenol by molybdopterin guanine dinucleotide released from dimethylsulfoxide reductase and by pterin standards was also measured and approximately 2 mol electrons/2 mol molybdopterin guanine dinucleotide were found to reduce 2,6-dichloroindophenol. These results are consistent with the presence of one molybdopterin guanine dinucleotide moiety with a pyrazine ring at the oxidation level of a dihydropteridine and one molybdopterin guanine dinucleotide moiety with a pyrazine ring at the oxidation level of a fully aromatic pteridine. It is suggested that the pyrazine ring of Q-molybdopterin guanine dinucleotide is fully aromatic and contains a 5,6 double bond.

Xanthine dehydrogenase, a molybdenum, iron-sulfur flavoenzyme encoded in the fruit fly Drosophila melanogaster by the rosy gene, has been characterised both from the wild-type and mutant files. Enzyme assays, using a variety of different oxidising and reducing substrates were supplemented by limited molecular characterisation. Four rosy strains showed no detectable activity in any enzyme assay tried, whereas from four wild-type and three rosy mutant strains, those for the [E89K], [L127F] and [L157P]xanthine dehydrogenases (in all of which the mutation is in the iron-sulfur domain), the enzyme molecules, although present at different levels, had extremely similar or identical properties. This was confirmed by purification of one wild-type and one mutant enzyme. [E89K]xanthine dehydrogenase. These both had ultraviolet-visible absorption spectra similar to milk xanthine oxidase. Both were found to be quite stable molecules, showing very high catalytic-centre activities and with little tendency to become degraded by proteolysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D]xanthine dehydrogenase and [S357F]xanthine dehydrogenase mutated in the flavin domain, and [G1011E]xanthine dehydrogenase mutated in the molybdenum domain, enzyme activities were selectively diminished in certain assays. For the G353D and S357F mutant enzymes activities to NAD+ as oxidising substrate were diminished, to zero for the latter. In addition for [G353D]xanthine dehydrogenase, there was an increase in apparent Km values both for NAD+ and NADH. These findings indicate involvement of this part of the sequence in the NAD(+)-binding site. The G1011E mutation has a profound effect on the enzyme. As isolated and as present in crude extracts of the files, this xanthine dehydrogenase variant lacks activity to xanthine or pterin as reducing substrate, indicating an impairment of the functioning of its molybdenum centre. However, it retains full activity to NADH with dyes as oxidising substrate. Mild oxidation of the enzyme converts it, apparently irreversibly, to a form showing full activity to xanthine and pterin. The nature of the group that is oxidised is discussed in the light of redox potential data. It is proposed that the process involves oxidation of the pterin of the molybdenum cofactor from the tetrahydro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Romao, M. J., Archer, M., Moura, I., Moura. J.J.G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270. 1170-1176) from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio gigas. It is proposed, that apparent irreversibility of the oxidative activating process for [G1011E]xanthine dehydrogenase, is due to conversion of its pterin to the tricyclic derivative detected by these workers. The data thus provide the strongest evidence available, that the oxidation state of the pterin can have a controlling influence on the activity of a molybdenum cofactor enzyme. Implications regarding pterin incorporation into xanthine dehydrogenase and in relation to other molybdenum enzymes are discussed.

Dimethylsulfide:acceptor oxidoreductase was purified from the purple non-sulfur phototrophic bacterium Rhodobacter sulfidophilus. The native form of the enzyme had a molecular mass of 152 kDa and was composed of three distinct subunits of 94, 38 and 32 kDa. Dimethylsulfide:acceptor oxidoreductase did not oxidise other thioethers which were tested. The enzyme was able to reduce a variety of N-oxides using reduced methylviologen as electron donor but it reduced dimethylsulfoxide at a very low rate. The resting form of dimethylsulfide:acceptor oxidoreductase exhibited a spectrum which was characteristic of a reduced cytochrome with absorbance maxima at 562 nm, 533 nm and 428 nm. Pyridine haemochrome analysis established that the cytochrome contained a b -type haem and a content of 0.65 mol protohaem/mol enzyme was determined. After oxidation of the haem with ferricyanide, the absorbance spectrum of the reduced cytochrome was restored by reduction with dimethylsulfide. Metal analysis revealed that dimethylsulfide:acceptor oxidoreductase contained 0.5 mol Mo and 3.5 mol Fe/mol enzyme. Heat treatment of the enzyme released material with fluorescence excitation and emission spectra which were characteristic of form B of the pterin component of the pterin molybdenum cofactor. From this analysis it is concluded that dimethylsulfide: acceptor oxidoreductase is a molybdenum oxotransferase which may also contain a iron-sulfur cluster. It is suggested that the haem and pterin molybdenum cofactor are associated with the 94-kDa subunit.

The molybdenum-containing iron-sulfur flavoprotein xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica has been purified approximately 800-fold with a yield of approximately 40% and a specific activity of approximately 70 micromol ferricyanide reduced x min(-1) x mg protein(-1) with xanthine as electron donor, which corresponds to approximately 30 micromol xanthine oxidized x min(-1) x mg protein(-1) with methylene blue as electron acceptor. The 129-kDa enzyme was a non-covalent heterotrimer with large (82.4 kDa), medium (28.5 kDa) and small (18.4 kDa) subunits. The N-termini of the small and medium polypeptides of V. atypica xanthine dehydrogenase and the corresponding domains of eukaryotic xanthine dehydrogenases were similar, whereas the N-terminus of the large polypeptide was unrelated to eukaryotic xanthine dehydrogenases. The enzyme contained 0.86 atoms Mo, 1.75 atoms Fe, 1.61 atoms acid-labile sulfur and 0.68 molecules FAD/molecule, which corresponds to a 1:2.0:1.9:0.8 molar ratio. Acid hydrolysis revealed 0.95 mol CMP and 0.80 mol AMP/mol xanthine dehydrogenase. After treatment of the enzyme with iodoacetamide, di(carboxamidomethyl)molybdopterin cytosine dinucleotide was identified, which indicates that molybdopterin cytosine dinucleotide is the organic portion of the V. atypica xanthine dehydrogenase molybdenum cofactor. The enzyme and its molybdenum cofactor occurred in a 1:1 molar ratio. Xanthine dehydrogenases from eukaryotic sources are characterized by a domain structure and the presence of duplicate copies of two types of [2Fe-2S) clusters. In contrast, the xanthine dehydrogenase from V. atypica had a heterotrimeric subunit structure and a single [2Fe-2S] cluster. In addition, the enzyme indicates the presence of a molybdopterin dinucleotide as a constituent of a xanthine dehydrogenase molybdenum cofactor.

Formylmethanofuran dehydrogenases are molybdenum or tungsten iron-sulfur proteins containing a pterin dinucleotide cofactor. We report here on the primary structures of the four subunits FwdABCD of the tungsten enzyme from Methanobacterium thermoautotrophicum which were determined by cloning and sequencing the encoding genes fwdABCD. FwdB was found to contain sequence motifs characteristic for molybdopterin-dinucleotide-containing enzymes indicating that this subunit harbors the active site. FwdA, FwdC and FwdD showed no significant sequence similarity to proteins in the data bases. Northern blot analysis revealed that the four fwd genes form a transcription unit together with three additional genes designated fwdE, fwdF and fwdG. A 17.8-kDa protein and an 8.6-kDa protein, both containing two [4Fe-4S] cluster binding motifs, were deduced from fwdE and fwdG. The open reading frame fwdF encodes a 38.6-kDa protein containing eight binding motifs for [4Fe-4S] clusters suggesting the gene product to be a novel polyferredoxin. All seven fwd genes were expressed in Escherichia coli yielding proteins of the expected size. The fwd operon was found to be located in a region of the M. thermoautotrophicum genome encoding molybdenum enzymes and proteins involved in molybdopterin biosynthesis.

A soluble alpha beta complex of nitrate reductase can be obtained from a strain of Escherichia coli that lacks the narI gene and expresses only the alpha and beta subunits. The beta subunit contains four Fe-S centres and the alpha subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Despite the lack of the gamma subunit of the complete enzyme, this complex can still catalyse the reduction of nitrate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre. To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenum cofactor, or both, we have studied the steady-state kinetics and the binding of benzyl viologen to the alpha beta complex and mutants alpha beta* with altered beta subunits. Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced. Two classes of site with different equilibrium constants could be distinguished. The kinetic results suggest that benzyl viologen supplies its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on the square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the alpha beta complex of nitrate reductase with artificial electron donors.

The molybdenum co-factor (Moco) is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes. This paper reports the isolation of cnx1, a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA. The mapping data of this cDNA correlate well with the mapping position of the A. thaliana molybdenum co-factor locus chl6. As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin. The protein encoded by cnx1 shows a two-domain structure: the N-terminal domain is homologous to the E. coli Moco protein MoeA, the C-terminal domain is homologous to the E. coli Moco proteins MoaB and MogA, respectively. These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E. coli, is performed by a single multifunctional protein in eukaryotes. In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis. These proteins also show a two-domain structure but the order of the domains is inversed as compared with Cnx1. Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.

A patient with molybdenum cofactor deficiency (producing the biochemical abnormalities associated with deficiencies of sulphite oxidase and xanthine dehydrogenase) clinically expressed Marfan-like habitus with dislocated lenses, vertebral abnormality, learning disability, moderate hemiplegia, increased medial lentiform MRI signal and intermittent microscopic haematuria.S-Sulphocysteine was present in plasma and urine, and the oxidized derivative of a molybdopterin precursor (precursor Z), together with xanthine and hypoxanthine, were elevated in urine. Blood uric acid was

Available mutants of molybdenum cofactor (MoCo) biosynthesis of Neurospora crassa were studied for converting factor activity and for in vitro molybdate repair of nitrate reductase (NR) activity. Mutant nit-7 was found to contain an activity that fits the functional definition of converting factor activity in Escherichia coli. Its high molecular weight fraction converts a low molecular weight compound from nit-1 and nit-8 into biologically active molybdopterin (MPT). Like nit-1, mutant nit-8 is devoid of this activity. Mutants nit-9 A, B and C contain a protein-bound precursor form of MoCo, which is presumed to be MPT bound to apo-NR. It is converted into active MoCo as part of NR in the presence of reduced glutathione and high exogenous molybdate concentrations. The NR apoenzyme of nit-1 is needed to detect the total amount of MoCo after molybdate repair, because mutants nit-9 A, B and C build no detectable content of functional NR apoenzyme. Evidence is presented for the transfer of MPT from demolybdo-NR to free NR apoenzyme.

Urothione was isolated as an excretion product of Hydrogenophaga pseudoflava and other bacteria at amounts approaching 253 micrograms/l of culture corresponding to 44 micrograms/g bacterial dry mass. The compound was identified as urothione by co-chromatography with urothione isolated from human urine, its characteristic ultraviolet and visible absorption spectra, oxidation to pterin-6-carboxylic-7-sulfonic acid by alkaline permanganate, 1H-NMR spectroscopy, double-quantum-filtered Fourier-transform 1H correlated spectroscopy, circular-dichroism spectroscopy and mass spectroscopy. A metabolic relationship between urothione and the carbon monoxide dehydrogenase molybdenum cofactor was suggested by a 1.1:0.5 molar ratio between urothione excreted and degradation of carbon monoxide dehydrogenase, a coincidence of urothione excretion and induction of carbon monoxide dehydrogenase with different species of carboxidotrophic bacteria, a structural relationship between molybdopterin cytosine dinucleotide of the carbon monoxide dehydrogenase molybdenum cofactor and urothione, and the demonstrated conversion of the carbon monoxide dehydrogenase molybdenum cofactor to urothione in vitro. A pathway for the conversion of the H. pseudoflava carbon monoxide dehydrogenase molybdenum cofactor to urothione has been proposed which involves molybdopterin cytosine dinucleotide, molybdopterin, phospho-norurothione and norurothione.

Sulphite oxidase deficiency occurs in man in two forms, as the isolated deficiency and as a syndrome of combined molybdoenzyme deficiency. This latter pleiotropic condition has as its underlying cause a defect in the synthesis of the molybdenum cofactor required for the activity of all molybdoenzymes in humans. Difficulties in diagnosis of sulphite oxidase deficiency are often encountered. A new method for detection of a key diagnostic metabolite, S-sulphocysteine, is outlined. The procedure is based on precolumn derivatization of urinary amino acids with dimethylaminoazobenzene sulphonyl chloride (Dabsyl-Cl) and resolution of the modified S-sulphocysteine by reversed-phase HPLC. A number of affected patients and control individuals with similar clinical symptoms have been studied, and a clear demarcation between the two groups has been noted.

Molybdenum cofactor deficiency is characterized by the absence of sulphite oxidase, xanthine dehydrogenase and aldehyde oxidase, the three known enzymes in man that require the cofactor for their activity. Prenatal diagnosis of the deficiency may be performed by assay of sulphite oxidase activity in cultured amniocytes. However, the activity in amniocytes is low and large numbers of cells are required for reliable assessment. We show that sulphite oxidase is present at high levels in chorionic villi obtained at 10-14 weeks gestation and can be assayed directly in the biopsy sample without cell culture. This assay has been applied to two pregnancies at risk for molybdenum cofactor deficiency with successful diagnoses of an unaffected and an affected fetus.

Formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum was purified to apparent homogeneity and found to contain per mol (apparent molecular mass 110 kDa) 0.6 mol molybdenum, 4 mol non-heme iron, 4 mol acid-labile sulfur, in addition, 0.7 mol of a pterin-containing co-factor (apparent molecular mass 800 Da) which has been characterized. The pterin material was extracted after alkylation by iodoacetamide and the extract subjected to HPLC on Lichrospher 100 RP-18. Three pterin compounds were resolved. On the basis of their UV/visible spectra and of the products formed after cleavage by nucleotide pyrophosphatase and alkaline phosphatase they were identified as the [di(carboxamidomethyl)]-derivatives of molybdopterin guanine dinucleotide (MGD) of molybdopterin adenine dinucleotide (MAD), and of molybdopterin hypoxanthine dinucleotide (MHD). The three pterin dinucleotides were present in the proportions 1:0,4:0.1.

The chlA locus encodes functions required for the biosynthesis of the molybdopterin part of the molybdenum cofactor. Mutants, carrying gene fusions at the chlA locus, which place beta-galactosidase expression under the control of the chlA promoter, have been isolated employing lambda placMu1 as the mutagen. The mutants exhibited beta-galactosidase expression which was greatly enhanced when grown anaerobically. Secondary mutations at the chlB, D, E or G loci did not affect the high level of expression. The fnr gene product was not required for the anaerobic expression. Bacteriophage lambda transducing phages were isolated which carried the phi(chlA-lac) mutations and were used to construct chlA+/phi(clA-lac) merodiploids. The merodiploids exhibited a much lower level of expression but showed the same characteristics as strains carrying lac fusions to the single chromosomal chlA locus. Genetic evidence is presented which strongly suggests that the molybdenum cofactor is a repressor of chlA expression. The anaerobic enhancement of chlA expression is mediated via a mechanism that is distinct from the molybdenum cofactor effect.

Molybdenum cofactor (MoCo) deficiency leads to a combined deficiency of the molybdo-enzymes sulphite oxidase, xanthine dehydrogenase and aldehyde oxidase. No therapy is known for this rare disease, which results in neonatal seizures and other neurological symptoms identical to sulphite oxidase deficiency. It is inherited autosomal-recessively and leads to early childhood death. Prenatal diagnosis has been performed since 1983 by the measurement of sulphite oxidase activity, but no enzymatic carrier diagnosis is possible. The human genes necessary for MoCo biosynthesis have recently been cloned and mutations in the bicistronic MOCS1 gene could be identified in most European patients. In a Danish family we have now performed enzymatic and molecular genetic analysis in parallel after chorionic villus sampling. The sulphite oxidase activity in uncultured CVS material was found to be normal. A MOCS1 splice site mutation, found homozygous in the affected patient, was found in a heterozygous state in cultured chorionic cells. This confirmed that the fetus was not affected, since heterozygous carriers of a MoCo deficiency allele do not display any symptoms.

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.

Molybdenum cofactor deficiency was diagnosed in a 3-month-old girl who presented with microcephaly, developmental delay, severe irritability, and lactic acidosis. Dietary methionine restriction, with cysteine supplementation, was associated with moderate short-term clinical improvement, including a resumption in predicted head growth, modest developmental progress, and a reduction in irritability. Clinical relapse was associated with noncompliance of dietary therapy 2 months later. Urinary sulfite levels measured by commercial dipsticks were useful in following therapy. Molybdenum cofactor deficiency is probably frequently underdiagnosed due to the lack of specific clinical or laboratory features. Screening of infants at risk for the presence of urinary sulfites or serum hypouricemia, or both, is both rapid and inexpensive.

A Chlamydomonas reinhardtii molybdenum cofactor (MoCo)-carrier protein (CP), capable of reconstituting nitrate reductase activity with apoprotein from the Neurospora crassa mutant nit-1, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unless aponitrate reductase was present in the incubation mixture, Stability of MoCo bound to CP against air and heat was very similar to that of free-MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme.

Five patients with a combined deficiency of xanthine dehydrogenase, sulphite oxidase and, possibly, also of aldehyde oxidase are described. This remarkable coincidence of three inborn errors of metabolism in a single individual was demonstrated to result from a deficiency of the 'molybdenum cofactor', an essential constituent of all three enzymes. The main biochemical findings in these patients included: hypouricaemia, xanthinuria, an increased excretion of sulphite, thiosulphate and S-SUL-sulphocysteine and a decreased excretion of inorganic sulphate. Plasma molybdenum was normal. The ultimate diagnosis was made by the measurement of 'molybdenum cofactor' in a liver biopsy specimen in three out of five patients. The clinical hallmarks in these patients were: feeding difficulties, mental retardation, neurological symptoms, lens dislocation, an abnormal muscle tone, myoclonia and an abnormal physiognomy. The majority of these were already present in the neonatal period. So far, attempts at treatment have been unsuccessful.

Radioactively labeled carbon monoxide (CO) dehydrogenase has been obtained in good yield and purity from Pseudomonas carboxydoflava grown in the presence of [32P]phosphate. One enzyme molecule contained an average of 8.32 molecules of phosphate. The entire phosphate content was confined to 2 molecules of FAD and 2 molecules of a pterin. These were noncovalently bound. Molybdoenzyme cofactors could be extracted into N-methyl formamide; pterins were isolated by thin-layer chromatography. CO dehydrogenase contained a novel pterin, different from molybdopterin, which was also resolved in other bacterial molybdoenzymes. Therefore, it was tentatively named bactopterin. The characteristic features of bactopterin were as follows. (a) A relative molecular mass, Mr, of 730 which was much greater than that of molybdopterin (330) (Mr values refer to molybdenum-free forms of the cofactors; presumably, the latter were also devoid of the sulfhydryl groups contained in the native compounds). (b) A content of 2 molecules of phosphate/molecule compared to only 1 phosphate in molybdopterin. (c) Bactopterin was three times less susceptible to air oxidation than molybdopterin. (d) Native bactopterin was cleaved by perchloric acid into two phosphorous-containing fragments with Mr, of 330 and 420. The smaller one is believed to be very similar to molybdopterin, the larger one was not a pterin but probably contained an aromatic structure.