Sulfite Impairs Bioenergetics and Redox Status in Neonatal Rat Brain: Insights into the Early Neuropathophysiology of Isolated Sulfite Oxidase and Molybdenum Cofactor Deficiencies.

Isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies are genetic diseases biochemically characterized by the toxic accumulation of sulfite in the tissues of patients, including the brain. Neurological dysfunction and brain abnormalities are commonly observed soon after birth, and some patients also have neuropathological alterations in the prenatal period (in utero). Thus, we investigated the effects of sulfite on redox and mitochondrial homeostasis, as well as signaling proteins in the cerebral cortex of rat pups. One-day-old Wistar rats received an intracerebroventricular administration of sulfite (0.5 µmol/g) or vehicle and were euthanized 30 min after injection. Sulfite administration decreased glutathione levels and glutathione S-transferase activity, and increased heme oxygenase-1 content in vivo in the cerebral cortex. Sulfite also reduced the activities of succinate dehydrogenase, creatine kinase, and respiratory chain complexes II and II-III. Furthermore, sulfite increased the cortical content of ERK1/2 and p38. These findings suggest that redox imbalance and bioenergetic impairment induced by sulfite in the brain are pathomechanisms that may contribute to the neuropathology of newborns with ISOD and MoCD. Sulfite disturbs antioxidant defenses, bioenergetics, and signaling pathways in the cerebral cortex of neonatal rats. CII: complex II; CII-III: complex II-III; CK: creatine kinase; GST: glutathione S-transferase; HO-1: heme oxygenase-1; SDH: succinate dehydrogenase; SO32-: sulfite.

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.

Site-Directed Mutagenesis at the Molybdenum Pterin Cofactor Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey.

The estimation of the drug clearance by aldehyde oxidase (AO) has been complicated because of this enzyme's atypical kinetics and species and substrate specificity. Since human AO (hAO) and cynomolgus monkey AO (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO, respectively, to compare and contrast the kinetics of these two species through numerical modeling. Whereas dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor to the corresponding alanine and leucine in mAO, respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, five more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. SIGNIFICANCE STATEMENT: Although they have as much as 95.1% sequence identity, human and cynomolgus monkey aldehyde oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans.

Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 µg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

Cloning and functional validation of molybdenum cofactor sulfurase gene from Ammopiptanthus nanus.

KEY MESSAGE: The molybdenum cofactor sulfurase gene ( AnMCSU ) was cloned from xerophytic desert plant Ammopiptanthus nanus and validated for its function of tolerance toward abiotic stresses by heterologous expression in Arabidopsis thaliana. Molybdenum cofactor sulfurase participates in catalyzing biosynthesis of abscisic acid, which plays a crucial role in the response of plants to abiotic stresses. In this study, we cloned molybdenum cofactor sulfurase gene (AnMCSU) from a super-xerophytic desert plant, Ammopiptanthus nanus, by using rapid amplification of cDNA ends method. This gene has a total length of 2544 bp, with a 5'- and a 3'-untranslated region of 167 and 88 bp, and an open reading frame of 2289 bp, which encodes an 84.85 kDa protein of 762 amino acids. The putative amino acid sequence shares high homology and conserved amino acid residues crucial for the function of molybdenum cofactor sulfurases with other leguminous species. The encoded protein of the AnMCSU gene was located in the cytoplasm by transient expression in Nicotiana benthamiana. The result of real-time quantitative PCR showed that the expression of the AnMCSU gene was induced by heat, dehydration, high salt stresses, and ABA induction, and inhibited by cold stress. The heterologous expression of the AnMCSU gene significantly enhanced the tolerance of Arabidopsis thaliana to high salt, cold, osmotic stresses, and abscisic acid induction. All these results suggest that the AnMCSU gene might play a crucial role in the adaptation of A. nanus to abiotic stress and has potential to be applied to transgenic improvement of commercial crops.

Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2.

Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.

Predicting intrinsic clearance for drugs and drug candidates metabolized by aldehyde oxidase.

Metabolism by aldehyde oxidase (AO) has been responsible for a number of drug failures in clinical trials. The main reason is the clearance values for drugs metabolized by AO are underestimated by allometric scaling from preclinical species. Furthermore, in vitro human data also underestimates clearance. We have developed the first in silico models to predict both in vitro and in vivo human intrinsic clearance for 8 drugs with just two chemical descriptors. These models explain a large amount of the variance in the data using two computational estimates of the electronic and steric features of the reaction. The in vivo computational models for human metabolism are better than in vitro preclinical animal testing at predicting human intrinsic clearance. Thus, it appears that AO is amenable to computational prediction of rates, which may be used to guide drug discovery, and predict pharmacokinetics for clinical trials.

Characterization of a mutant of Chlamydomonas reinhardtii deficient in the molybdenum cofactor.

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.

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.

Convergent evolution of hydroxylation mechanisms in the fungal kingdom: molybdenum cofactor-independent hydroxylation of xanthine via alpha-ketoglutarate-dependent dioxygenases.

The xanthine oxidases and dehydrogenases are among the most conserved enzymes in all living kingdoms. They contain the molybdopterin cofactor Moco. We show here that in the fungi, in addition to xanthine dehydrogenase, a completely different enzyme is able to catalyse the oxidation of xanthine to uric acid. In Aspergillus nidulans this enzyme is coded by the xanA gene. We have cloned the xanA gene and determined its sequence. A deletion of the gene has the same phenotype as the previously known xanA1 miss-sense mutation. Homologues of xanA exist only in the fungal kingdom. We have inactivated the cognate gene of Schizosaccharomyces pombe and this results in strongly impaired xanthine utilization as a nitrogen source. We have shown that the Neurospora crassa homologue is functionally equivalent to xanA. The enzyme coded by xanA is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase which shares a number of properties with other enzymes of this group. This work shows that only in the fungal kingdom, an alternative mechanism of xanthine oxidation, not involving Moco, has evolved using the dioxygenase scaffold.

The pathogenesis of molybdenum cofactor deficiency, its delay by maternal clearance, and its expression pattern in microarray analysis.

Molybdenum cofactor (Moco)-deficiency is a lethal autosomal recessive disease, for which until now no effective therapy is available. The biochemical hallmark of this disorder is the inactivity of the Moco-dependent sulfite oxidase, which results in elevated sulfite and diminished sulfate levels throughout the organism. In humans, Moco-deficiency results in neurological damage, which is apparent in untreatable seizures and various brain dysmorphisms. We have recently described a murine model for Moco-deficiency, which reflects all enzyme and metabolite changes observed in the patients, and an efficient therapy using a biosynthetic precursor of Moco has been established in this animal model. We now analyzed these mice in detail and excluded morphological brain damage, while expression analysis with microarrays indicates a massive cell death program. This neuronal damage appears to be triggered by elevated sulfite levels and is ameliorated in affected embryos by maternal clearance.

Mechanistic studies of human molybdopterin synthase reaction and characterization of mutants identified in group B patients of molybdenum cofactor deficiency.

Biosynthesis of the molybdenum cofactor involves the initial formation of precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. Characterization of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of precursor Z.

Mcp1 encodes the molybdenum cofactor carrier protein in Chlamydomonas reinhardtii and participates in protection, binding, and storage functions of the cofactor.

The molybdenum cofactor (Moco) is essential for the activity of all molybdoenzymes except nitrogenase. The cDNA for the Moco carrier protein (MocoCP) of Chlamydomonas reinhardtii has been cloned by reverse transcription PCR approaches with primers designed from microsequenced peptides of this protein. The C. reinhardtii MocoCP has been expressed in Escherichia coli. The recombinant protein has been purified to electrophoretic homogeneity and is found assembled into a homotetramer when Moco is not present under native conditions. Recombinant MocoCP has the same biochemical characteristics as MocoCP from C. reinhardtii, as it bound Moco from milk xanthine oxidase with high affinity, prevented Moco inactivation by oxygen, and transferred Moco efficiently to aponitrate reductase from the Neurospora crassa nit1 mutant. The genomic DNA sequence corresponding to the Chlamydomonas MocoCP gene, CrMcp1, also was isolated. This gene contained three introns in the coding region. The deduced amino acid sequence of CrMcp1 did not show a significant identity to functionally known proteins in the GenBank data base, although a significant conservation was found with bacterial proteins of unknown function. The results suggest that proteins having a Moco binding function probably exist in other organisms.

Catalysis at a dinuclear [CuSMo(==O)OH] cluster in a CO dehydrogenase resolved at 1.1-A resolution.

The CO dehydrogenase of the eubacterium Oligotropha carboxidovorans is a 277-kDa Mo- and Cu-containing iron-sulfur flavoprotein. Here, the enzyme's active site in the oxidized or reduced state, after inactivation with potassium cyanide or with n-butylisocyanide bound to the active site, has been reinvestigated by multiple wavelength anomalous dispersion measurements at atomic resolution, electron spin resonance spectroscopy, and chemical analyses. We present evidence for a dinuclear heterometal [CuSMoO)OH] cluster in the active site of the oxidized or reduced enzyme, which is prone to cyanolysis. The cluster is coordinated through interactions of the Mo with the dithiolate pyran ring of molybdopterin cytosine dinucleotide and of the Cu with the Sgamma of Cys-388, which is part of the active-site loop VAYRC(388)SFR. The previously reported active-site structure [Dobbek, H., Gremer, L., Meyer, O. & Huber, R. (1999) Proc. Natl. Acad. Sci. USA 96, 8884-8889] of an Mo with three oxygen ligands and an SeH-group bound to the Sgamma atom of Cys-388 could not be confirmed. The structure of CO dehydrogenase with the inhibitor n-butylisocyanide bound has led to a model for the catalytic mechanism of CO oxidation which involves a thiocarbonate-like intermediate state. The dinuclear [CuSMo(O)OH] cluster of CO dehydrogenase establishes a previously uncharacterized class of dinuclear molybdoenzymes containing the pterin cofactor.

Molybdopterin synthase mutations in a mild case of molybdenum cofactor deficiency.

Molybdenum cofactor deficiency is a rare inborn error of metabolism with generally severe symptoms, most often including neonatal seizures and severe developmental delay. We describe a patient with an unusually mild form of the disease. Two mutations in MOCS2A (molybdenum cofactor synthesis enzyme 2A) were identified: a single base change, 16C > T, that predicts a Q6X substitution on one allele and a 19G > T transversion that predicts a valine to phenylalanine substitution, V7F, on the second. It is postulated that the milder clinical symptoms result from a low level of residual molybdopterin synthase activity derived from the 19G > T allele.

Deletion of the cnxE gene encoding the gephyrin-like protein involved in the final stages of molybdenum cofactor biosynthesis in Aspergillus nidulans.

The Aspergillus nidulans cnxE gene, required for molybdenum cofactor biosynthesis, was isolated by functional complementation of an Escherichia coli mogA mutant strain. The deduced CnxE polypeptide consists of two domains which display similarity to the E. coli proteins MoeA and MogA, respectively, separated by a putative hinge region of around 58 amino acid residues which is notably histidine rich. A deletion mutant lacking the entire cnxE gene, including both MoeA-like and MogA-like domains, was identified. Compared to the wild type, a small increase in the intermediate precursor Z was observed in the deletion strain but was significant only under conditions in which the molybdoenzyme nitrate reductase was induced. Elevated levels of the pathway intermediate molybdopterin were found both under nitrate reductase-inducing and non-inducing conditions in the deletion mutant compared to the wild type. This increase is in contrast to previous results for cnxABC, cnxF, cnxG, and cnxH mutants, in which the levels of molybdopterin were substantially reduced, and therefore supports previously published classical genetic and biochemical studies that indicated that the CnxE protein is likely to be involved in the final stages of molybdenum cofactor biosynthesis. We have found no evidence during our chemical analysis for any involvement of this protein in the intermediate section of the molybdenum cofactor biosynthetic pathway (i.e. in the synthesis of molybdopterin from precursor Z), as has been suggested previously for E. coli MoeA. The 2.5-kb cnxE transcript is not abundant and appears to be expressed constitutively.

Leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum.

We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S(1) nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from -50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the -1 position and a BRE element with more frequent A bases at position -29 to -32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.