Molybdenum-containing hydroxylases

Unlike monooxygenases, molybdenum-containing hydroxylases catalyze the hydroxylation of carbon centers using oxygen derived ultimately from water, rather than O(2), as the source of the oxygen atom incorporated into the product, and do not require an external source of reducing equivalents. The mechanism by which this interesting chemistry takes place has been the subject of investigation for some time, and in the last several years the chemical course of the reaction has become increasingly well understood. The present minireview summarizes recent mechanistic and structure/function studies of members of this large and growing family of enzymes.     

高等植物中的钼

阐述了高等植物缺钼症状,分析了植物体存在的钼酥及钼辅因子的生理生化特性和生理功能及钼辅因子的可能合成途径,并提出以后的研究方向。     

Effect of Beer Ingestion on the Plasma Concentrations and Urinary Excretion of Purine Bases: One-Month Study

To investigate the effect of long-term beer ingestion on the plasma concentrations and urinary excretion of purine bases, 5 healthy males participated in the present study, during which they ingested beer every evening for 30 days. Blood and 24-hour urine samples were collected in the morning one day before and 14 and 30 days after the initiation of the beer ingestion. During the beer ingestion period, the plasma concentration and the urinary excretion of uric acid were increased significantly, while uric acid clearance was not decreased. Further, purine ingestion was not significantly different throughout the study. These results suggest that production of uric acid by ethanol ingestion was the main contributor to the increased plasma uric acid. Therefore, patients with gout should be encouraged to avoid drinking large amounts of beer on a daily basis.     

Characterization of a methyl-specific restriction system in Clostridium acetobutylicum strain N1-4081

A type II restriction endonuclease, named CacI, was detected in Clostridium acetobutylicum strain N1-4081. CacI cleaved the tetranucleotide sequence [5' decreases -GATC-3']. The modification system consisted of the methylation of the adenine present in this sequence. CacI, an isoschizomer of MboI, is inactive on dam methylated substrates.     

The chaperone FdsC for Rhodobacter capsulatus formate dehydrogenase binds the bis-molybdopterin guanine dinucleotide cofactor

Molybdoenzymes are complex enzymes in which the molybdenum cofactor (Moco) is deeply buried in the enzyme. Most molybdoenzymes contain a specific chaperone for the insertion of Moco. For the formate dehydrogenase FdsGBA from Rhodobacter capsulatus the two chaperones FdsC and FdsD were identified to be essential for enzyme activity, but are not a subunit of the mature enzyme. Here, we purified and characterized the FdsC protein after heterologous expression in Escherichia coli. We were able to copurify FdsC with the bound Moco derivate bis-molybdopterin guanine dinucleotide. This cofactor successfully was used as a source to reconstitute the activity of molybdoenzymes.     

Tissue-specific regulation of rice molybdenum cofactor sulfurase gene in response to salt stress and ABA

Molybdenum-containing aldehyde oxidase is a key enzyme for catalyzing the final step of abscisic acid (ABA) biosynthesis in plants. Sulfuration of the molybdenum cofactor (MoCo) is an essential step for activating aldehyde oxidase. The molybdenum cofactor sulfurase (MCSU) that transfers the sulfur ligand to aldehyde oxidase-bound MoCo is thus considered an important factor in regulating the ABA levels in plant tissues. In this study, we identified the rice MCSU cDNA (OsMCSU), which is the first MCSU gene cloned in monocot species. According to the functional domain analysis of the predicted amino acid sequence, the OsMCSU protein contains a Nifs domain at its N-terminus and a MOSC domain at the C-terminus. Expression of the OsMCSU gene was up-regulated by salt stress in root tissues of rice seedlings, but this effect was not observed in leaf tissues. In roots, regulations of OsMCSU expressions could be mediated by both ABA-dependent and ABA-independent signaling pathways under salt stress condition.     

Xanthine and Uric Acid Levels in Rat Brain Following Focal Ischemia

Changes of the xanthine and uric acid (UA) levels in rat forebrain following focal cerebral ischemia were studied by reversed-phase HPLC with electrochemical detection. Focal ischemia was induced by occluding the left middle cerebral artery in the rat. The xanthine level in the normal group was 11.50 nmol/g tissue. In the ischemic group, the xanthine concentration in the ischemic hemisphere progressively increased after occlusion and reached a maximum value of 59.42 nmol/g tissue 4 h after operation. The UA level in the normal group was 2.20 nmol/g tissue, whereas in the ischemic group the UA concentration in the ischemic hemisphere gradually increased after occlusion, reaching a value of 38.53 nmol/g tissue 24 h after ischemia. The concentration of UA remained elevated in the ischemic hemisphere until 48 h after occlusion, and reached a maximum value of 38.98 nmol/g tissue. The xanthine and UA levels in the contralateral hemisphere remained unchanged. The xanthine and UA concentrations in the sham-operated group did not show a significant increase after operation. The time course of xanthine and UA levels suggests that in ischemic forebrain UA is formed from xanthine as a product of purine metabolism.     

Redox reactions of the pyranopterin system of the molybdenum cofactor

This work provides the first extensive study of the redox reactivity of the pyranopterin system that is a component of the catalytic site of all molybdenum and tungsten enzymes possessing molybdopterin. The pyranopterin system possesses certain characteristics typical of tetrahydropterins, such as a reduced pyrazine ring; however, it behaves as a dihydropterin in redox reactions with oxidants. Titrations using ferricyanide and dichloroindophenol (DCIP) prove a 2e^–/2H⁺ stoichiometry for pyranopterin oxidations. Oxidations of pyranopterin by Fe(CN)₆ ^3– or DCIP are slower than tetrahydropterin oxidation under a variety of conditions, but are considerably faster than observed for oxidations of dihydropterin. The rate of pyranopterin oxidation by DCIP was studied in a variety of media. In aqueous buffered solution the pyranopterin oxidation rate has minimal pH dependence, whereas the rate of tetrahydropterin oxidation decreases 100-fold over the pH range 7.4–8.5. Although pyranopterin reacts as a dihydropterin with oxidants, it resists further reduction to a tetrahydropterin. No reduction was achieved by catalytic hydrogenation, even after several days. The reducing ability of the commonly used biological reductants dithionite and methyl viologen radical cation was investigated, but experiments showed no evidence of pyranopterin reduction by any of these reducing agents. This study illustrates the dual personalities of pyranopterin and underscores the unique place that the pyranopterin system holds in the spectrum of pterin redox reactions. The work presented here has important implications for understanding the biosynthesis and reaction chemistry of the pyranopterin cofactor in molybdenum and tungsten enzymes.Abbreviations DCIP dichloroindophenol - H₄DMP 6,7-dimethyltetrahydropterin - MV⁺ methyl viologen radical cation     

Efficacy and Safety of Allopurinol in Patients with the Lesch-Nyhan Syndrome and Partial Hypoxanthine- Phosphoribosyltransferase Deficiency: a Follow-up Study of 18 Spanish Patients

Allopurinol is used widely for the treatment of purine disorders such as gout, but efficacy and safety of allopurinol has not been analyzed systematically in an extensive series of patients with HPRT deficiency. From 1984 to 2004 we have diagnosed 30 patients with HPRT deficiency. Eighteen patients (12 with Lesch-Nyhan syndrome or complete HPRT deficiency, and 6 with partial HPRT deficiency) were treated with allopurinol (mean dose, 6.44 mg/Kg of weight per day) and followed-up for at least 12 months (mean follow-up 7,6 years per patient). Mean age at diagnosis was 7 years (range, 5 months to 35 years). Treatment with allopurinol was associated to a mean reduction of serum urate concentration of 50%, and was normalized in all patients. Mean urinary uric acid excretion was reduced by 75% from baseline values, and uric acid to creatinine ratio was close or under 1.0 in all patients. In contrast, hypoxanthine and xanthine urinary excretion rates increased by a mean of 6 and 10 times, respectively, compared to baseline levels. These modifications were similar in patients with complete or partial HPRT deficiency. In 2 patients xanthine stones were documented despite allopurinol dose adjustments to prevent markedly increased oxypurine excretion rates. Neurological manifestations did not appear to be influenced by allopurinol therapy. Allopurinol is a very efficacy and fairly safety drug for the treatment of uric acid overproduction in patients with complete and partial HPRT deficiency. Allopurinol was associated with xanthine lithiasis.     

Molybdenum-dependent degradation of nicotinic acid by Bacillus sp. DSM 2923

Abstract Growth of Bacillus sp. DSM 2923 on nicotinic acid in mineral medium was dependent on the concentration of sodium molybdate added. Addition of increasing amounts of tungstate to the medium resulted in an inhibition of growth on nicotinic acid or 6-hydroxynicotinic acid as sole source of carbon and energy. Chlorate-resistant mutants were isolated which were not able to degrade nicotinic acid and 6-hydroxynicotinic acid nor to reduce nitrate. Additionally, enzyme activities of nicotinic acid dehydrogenase and 6-hydroxynicotinic acid dehydrogenase increased with increasing concentrations of molybdate (10⁻⁸ to 10⁻⁶ M) added to the medium, and decreased with increasing amounts of tungstate (10⁻⁶ to 10⁻⁵ M) in the medium.     

Peroxisomal xanthine oxidoreductase: characterization of the enzyme from pea (Pisum sativum L.) leaves

The presence and properties of the enzyme xanthine oxidoreductase (XOR) in peroxisomes from pea (Pisum sativum L.) leaves were studied using biochemical and immunological methods. The activity analysis showed that, in leaf peroxisomes, the superoxide-generating XOR form, xanthine oxidase (XOD), was predominant over the xanthine dehydrogenase form (XDH), with a XDH/XOD ratio of 0.5. However, in crude extracts of pea leaves, the XDH form was more abundant, with a XDH/XOD ratio of 1.6. The native molecular mass of the peroxisomal XOR determined by polyacrylamide gel electrophoresis was 290kDa. Using western blot assays, we identified an immunoreactive band of 59kDa that was not affected by the reducing reagent DTT or endogenous proteases. The analysis of pea leaves by electron microscopy immunogold labeling with affinity-purified antibodies against rat XOD confirmed that this enzyme was localized in the matrix of peroxisomes, as well as in chloroplasts and cytosol. In pea plants subjected to abiotic stress by cadmium, the activity of the peroxisomal XOR was reduced, whereas its protein level expression increased. The results confirmed that leaf peroxisomes contain XOR, and suggest that this peroxisomal metalloflavoprotein enzyme is involved in the mechanism of response of pea plants to abiotic stress by heavy metals.     

A Turkish Case with Molybdenum Cofactor Deficiency

Molybdenum cofactor deficiency (MIM 252150) is a rare progressive neurodegenerative disorder with about 100 cases reported worldwide. We have identified a male with molybdenum cofactor deficiency and analyzed the molybdenum cofactor synthesis (MOCS)1 gene, MOCS2 gene, MOCS3 gene and GEPH gene. We homozygously identified the CGA insertion after A666 of the MOCS1 gene which produces arginine insertion at codon 222 of MOCS1A. The parents, his brother and his sister who did not have any symptoms were heterozygous for the same mutation. This region was highly conserved in various species. The N-terminal part of MOCS1 a protein is suggested to form the central core of the protein and be composed of an incomplete [(alpha/beta)6] triosephosphate isomerase (TIM) barrel with a lateral opening that is covered by the C-terminal part of the protein. The insertion is located in the loop connecting the fifth beta strand to the sixth alpha helices of the TIM barrel structure. This arginine insertion would induce the conformation change and the lack of the activity.     

RNA interference-mediated suppression of xanthine dehydrogenase reveals the role of purine metabolism in drought tolerance in Arabidopsis

We have previously demonstrated that RNA interference-mediated suppression of xanthine dehydrogenase (XDH), the rate-limiting enzyme in purine degradation, causes defects in the normal growth and development of Arabidopsis thaliana. Here, we investigated a possible role for XDH in drought tolerance, since this enzyme is also implicated in plant stress responses and acclimatization. When XDH-suppressed lines were subjected to drought stress, plant growth was markedly reduced in conjunction with significantly enhanced cell death and H₂O₂ accumulation. This drought-hypersensitive phenotype was reversed by pretreatment with exogenous uric acid, the catalytic product of XDH. These results suggest that fully functional purine metabolism plays a role in the Arabidopsis drought acclimatization.     

Effect of valproic acid on the urinary metabolic profile of a patient with succinic semialdehyde dehydrogenase deficiency

The metabolic changes in a patient with succinic semialdehyde dehydrogenase deficiency were investigated following valproate administration using urease pretreatment and gas chromatography-mass spectrometry. A stable isotope dilution technique was used for quantification of urinary 4-hydroxybutyrate. Urinary levels of 4-hydroxybutyrate were 4-fold higher after 1-month valproate therapy. 4,5-Dihydrohexanoate, 2-deoxytetronate and 3-deoxytetronate were also 1.7-2.7-fold higher. The urinary excretions of 4-hydroxybutyrate in valproate non-medicated controls were age dependence and decreased with age. Relationships between 4-hydroxybutyrate excretion and 4-hydroxyvalproate or 5-hydroxyvalproate excretion were observed in valproate medicated controls. It seems that 4-hydroxyvalproate and 5-hydroxyvalproate as well as valproate are involved with increased excretion of 4-hydroxybutyrate following valproate administrations.     

Increased Uric Acid in the Developing Brain and Spinal Cord Following Cytomegalovirus Infection

Abstract: Tissue concentrations of uric acid were determined in the spinal cord, cerebellum, caudate-putamen, and cerebral cortex of developing mice following intraventricular inoculation with murine cytomegalovirus (MCMV) on postnatal day 10. Transient signs of neurological impairment were observed in MCMV-infected animals beginning on days 13–16 and continuing until days 19–21. At the onset of neurological impairment, uric acid concentrations in tissues from infected animals were 17–60-fold greater than in control animals. On postnatal day 70, 60 days after inoculation and 40 days after resolution of neurological signs, uric acid levels were still two- to threefold greater in infected animals. Histological examination revealed signs of focal ischemia in the cerebral and cerebellar cortices of MCMV-infected mice only at the onset of neurological impairment, with ischemic cell changes in some pyramidal neurons of the cerebral cortex. These results indicate that uric acid may be a sensitive marker of persistent vascular pathology resulting from cytomegalovirus infection of the developing nervous system     

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O₂ ^•− source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O₂ ^•− molecule and half a H₂O₂ molecule per NADH molecule, at rates 3 times those observed for XO (29.2 ± 1.6 and 9.38 ± 0.31 min⁻¹, respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 ± 1.36 μM⁻¹ min⁻¹) was found to be higher than that of the XO specificity constant (1.07 ± 0.09 μM⁻¹ min⁻¹). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O₂ ^•− source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O₂ ^•− than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.