The pterin molybdenum cofactors.
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Coenzymes↗
Biomedical subjects
K V Rajagopalan
Publications and source records attributed to K V Rajagopalan.
Proposed nomenclature for the genes involved in molybdenum metabolism in Escherichia coli and Salmonella typhimurium.
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Coenzymes↗
31P-NMR of free and protein-bound molybdopterin guanine dinucleotide.
Molybdopterin guanine dinucleotide was studied by 31P-NMR in the free, iodoacetamide derivatized form [di(carboxamidomethyl)molybdopterin] and in the native state in the dimethyl sulfoxide reductase from Rhodobacter sphaeroides. The spectra confirm the presence of a pyrophosphate moiety in the cofactor molecule. Comparison of the spectrum of the free pterin with that of the protein-bound cofactor reveals a substantial upfield shift of the 31P resonances in the enzyme-bound form with respect to the free form. This shift is attributed to differences in the bond and torsional angles of the phosphates. The spectrum of the protein suggests significant coupling between the two phosphorus nuclei with coupling constants of approximately 200 Hz. Comparison of the 31P-NMR spectra of molybdopterin guanine dinucleotide and flavin adenine dinucleotide suggests that the two cofactors have similar conformations in both their free and protein-bound forms.
Animals↗
Rat sulfite oxidase antibodies cross-react with two gene family-related proteins: albumin and vitamin D-binding protein.
Screening lambda cDNA libraries from rat liver with antibody to native rat liver sulfite oxidase (RLSO) showed cross-reaction with two proteins that belong to the same gene family: serum albumin and vitamin D-binding protein. Antibodies raised against native RLSO or sodium dodecyl sulfate-denatured protein cross-reacted with these proteins by Western blot analysis. The relative effectiveness of RLSO antibody binding was estimated to be 1/5 for rat serum albumin and 1/10 for rat vitamin D-binding protein. This result was not caused by contaminating proteins in the RLSO used for immunization as the RLSO preparation did not react with rat serum albumin antibody. RLSO antibodies, selected for their ability to bind rat serum albumin immobilized on nitrocellulose, recognized both rat serum albumin and RLSO. RLSO antibody, with albumin-reactive antibody removed, still recognized vitamin D-binding protein, suggesting that multiple determinants specific to each protein are involved in the cross-reaction. Comparison of RLSO antibody binding to the rat and human proteins indicated that the determinants were species-specific. cDNA clones identified by screening cDNA libraries with RLSO antibody demonstrated that these determinants reside in the C-terminal domain of these proteins. These results suggest that these proteins contain some common immunological features and may be evolutionarily related.
Albumins↗
The mechanisms of inactivation of sulfite oxidase by periodate and arsenite.
The inactivation of sulfite oxidase, a molybdoenzyme containing the Mo cofactor, by arsenite and periodate was investigated. In contrast to ferricyanide (Gardlik, S., and Rajagopalan, K.V. (1991) J. Biol. Chem. 266, 4889-4895), neither of these reagents causes oxidation of the pterin ring of the Mo cofactor. Instead, inactivation by these reagents appears to involve attack on sulfhydryl groups at the active site of the enzyme. The inactivation of sulfite oxidase by arsenite was shown to be dependent on the presence of O2 and on the enzymatic oxidation of arsenite to arsenate. The inactivation was preventable by the presence of sulfite, or by the use of cytochrome c as the electron acceptor instead of O2. It is concluded that inactivation by arsenite is the result of arsenite displacement of Mo during enzymatic oxidation of arsenite to arsenate, when Mo transiently breaks its bond to protein or molybdopterin sulfhydryl(s) in order to provide a site for transfer of electrons to O2. Data indicate that arsenite is properly oriented to displace Mo only once every 20,800 turnovers, thus accounting for the slow rate of inactivation by this reagent. Inactivation of sulfite oxidase by periodate is believed to occur as the result of direct attack of periodate on the thiolate ligands of Mo, either those of the protein and/or molybdopterin, leading to Mo loss. Treatment of enzyme with even low levels of periodate resulted in loss of Mo and both sulfite:cytochrome c and sulfite:O2 activities. Molybdopterin of periodate-inactivated enzyme retained the ability to reconstitute nitrate reductase apoprotein in nit-1 extracts and the ability to reduce dichlorophenolindophenol, indicating that the pterin ring had not been oxidized.
Animals↗
Molybdenum cofactor biosynthesis in Escherichia coli. Requirement of the chlB gene product for the formation of molybdopterin guanine dinucleotide.
The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor. The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin. The molybdenum cofactor in a number of E. coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain. In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E. coli. Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E. coli cells. Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide.
Chromatography, High Pressure Liquid↗
Oxidation of molybdopterin in sulfite oxidase by ferricyanide. Effect on electron transfer activities.
The attenuation of the sulfite:cytochrome c activity of sulfite oxidase upon treatment with ferricyanide was demonstrated to be the result of oxidation of the pterin ring of the molybdenum cofactor in the enzyme. Oxidation of molybdopterin (MPT) was detected in several ways. Ferricyanide treatment not only abolished the ability of sulfite oxidase to serve as a source of MPT to reconstitute the aponitrate reductase in extracts of the Neurospora crassa mutant nit-1 but also eliminated the ability of sulfite oxidase to reduce dichlorobenzenoneindophenol after anaerobic denaturation. Additionally, the absorption spectrum of anaerobically denatured ferricyanide-treated molybdenum fragment of rat liver sulfite oxidase was typical of fully oxidized pterins. Ferricyanide treatment had no effect on the protein of sulfite oxidase or on the sulfhydryl-containing side chain of MPT. Quantitation of the ferricyanide reaction showed that 2 mol of ferricyanide were reduced per mol of MPT oxidized, yielding a fully oxidized pterin. These results corroborate the previously reported conclusion that the native state of reduction of MPT in sulfite oxidase is at the dihydro level (Gardlik, S., and Rajagopalan, K.V. (1990) J. Biol. Chem. 265, 13047-13054). As a result of oxidation of the pterin ring, the affinity of MPT for molybdenum is decreased, leading to eventual loss of molybdenum. Because the loss of molybdenum is slow, a population of sulfite oxidase molecules can exist in which molybdenum is complexed to oxidized MPT. These molecules retain sulfite:O2 activity, a function apparently dependent solely on the molybdenum-thiolate complex, yet have greatly decreased sulfite:cytochrome c activity, a function requiring heme as well as the molybdenum center of holoenzyme. These observations suggest that the pterin ring of MPT participates in enzyme function, possibly in electron transfer, directly in catalysis, or by controlling the oxidation/reduction potential of molybdenum.
Animals↗
Identification of molybdopterin guanine dinucleotide in formate dehydrogenase from Methanobacterium formicicum.
The pterin cofactor in formate dehydrogenase isolated from Methanobacterium formicium is identified as molybdopterin guanine dinucleotide. The pterin, stabilized as the alkylated, dicarboxamidomethyl derivative, is shown to have absorption and chromatographic properties identical to those of the previously characterized authentic compound. Treatment with nucleotide pyrophosphatase produced the expected degradation products GMP and carboxyamidomethyl molybdopterin. The molybdopterin guanine dinucleotide released from the enzyme by treatment with 95% dimethyl sulfoxide is shown to be functional in the in vitro reconstitution of the cofactor-deficient nitrate reductase in extracts of the Neurospora crassa nit-1 mutant.
Chromatography, High Pressure Liquid↗
Spectroscopic studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans.
Absorption and EPR spectroscopic properties of purified dimethyl sulfoxide (Me2SO) reductase from Rhodobacter sphaeroides f. sp. denitrificans have been examined. The absence of prosthetic groups other than the molybdenum center in the enzyme has made it possible to study its absorption properties. The enzyme displays multiple absorbance peaks in both the oxidized and the dithionite-reduced forms. The oxidized enzyme has absorbance peaks at 280, 350, 470, 550, and 720 nm while the dithionite-reduced enzyme has peaks at 280, 374, and 645 nm with a shoulder at 430 nm. A comparison of the absorbance spectrum of oxidized Me2SO reductase with that of the molybdenum fragment of rat liver sulfite oxidase shows that the 350 and 470 peaks are common to both proteins. EPR studies of the Mo(V) form of Me2SO reductase show a rhombic signal with g1 = 1.988, g2 = 1.977, g3 = 1.961, and g(ave) = 1.975. The signal shows evidence of coupling to an exchangeable proton with A1 = 1.05, A2 = 1.13, A3 = 0.98, and Aave = 1.05 millitesla. These parameters are similar to those of other Mo enzymes, however, the epr signal of this enzyme differs from those of other Mo hydroxylases in showing only a slight sensitivity to pH and no detectable anion effect. EPR potentiometric titrations of Me2SO reductase gave midpoint potentials of +144 mV for the Mo(VI)/Mo(V) couple and +160 mV for the Mo(V)/Mo(IV) couple at room temperature and +141 mV for the Mo(VI)/Mo(V) couple and +200 mV for the Mo(V)/Mo(IV) couple at 173 K.
Chromatography, Ion Exchange↗
Novel aspects of the biochemistry of the molybdenum cofactor.
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Animals↗
Prenatal diagnosis of molybdenum cofactor deficiency by assay of sulphite oxidase activity in chorionic villus samples.
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.
Aldehyde Oxidase↗
Identification of a molybdopterin-containing molybdenum cofactor in xanthine dehydrogenase from Pseudomonas aeruginosa.
Xanthine dehydrogenase has been purified from Pseudomonas aeruginosa cultured on a rich medium and induced with hypoxanthine. The enzyme was shown to contain FAD, iron sulfur centers and a molybdenum cofactor as prosthetic groups. Analysis of the molybdenum cofactor in this enzyme has revealed that the cofactor contains molybdopterin (MPT) rather than molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide which have previously been identified in a number of molybdoenzymes of bacterial origin. The pterin cofactor in P.aeruginosa xanthine dehydrogenase was alkylated and the resulting product was identified as dicarboxamidomethyl molybdopterin. In addition, the pterin released from the enzyme by denaturation with guanidine-HCl was found to chromatograph on Sephadex G-15 with an apparent molecular weight of 350. These results document the first example of a bacterial enzyme with a molybdenum cofactor comprising molybdopterin and the metal only.
Animals↗
The folate cofactor of Escherichia coli DNA photolyase acts catalytically.
Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.
Catalysis↗
The state of reduction of molybdopterin in xanthine oxidase and sulfite oxidase.
Methods have been devised to examine the spectral properties and state of reduction of the pterin ring of molybdopterin (MPT) in milk xanthine oxidase and the Mo-containing domain of rat liver sulfite oxidase. The absorption spectrum of the native pterin was visualized by difference spectroscopy of each protein, denatured anaerobically in 6 M guanidine hydrochloride (GdnHCl), versus a sample containing the respective apoprotein and other necessary components. The state of reduction of MPT was also probed using 2,6-dichlorobenzenoneindophenol (DCIP) to measure reducing equivalents/MPT, after anaerobic denaturation of the protein in GdnHCl in the presence or absence of Hg2+. In the case of xanthine oxidase the data indicate that the terminal sulfide ligand of Mo causes the reduction of a native dihydro form of MPT to the tetrahydro level. This reduction does not occur if Hg2+ is added prior to denaturation of the protein. Based on its observed behavior, the native MPT in the Mo cofactor of xanthine oxidase is postulated to exist as a quinonoid dihydropterin. Quantitation of DCIP reduction by MPT of Mo fragment of sulfite oxidase showed a two-electron oxidation of MPT, even when the Mo fragment was denatured in the presence of Hg2+ to prevent internal reduction reactions due to sulfhydryls or sulfide. Difference spectra of DCIP-treated versus untreated Mo fragment showed that MPT had been fully oxidized. These data indicate that the native MPT in sulfite oxidase must be a dihydro isomer different from that in xanthine oxidase.
Animals↗
The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants.
Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E. coli strains lacking dihydrofolate reductase. The extent of over-production in the mutants was comparable to that seen in the wild type strain. Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain. Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives. These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E. coli.
Chromatography, High Pressure Liquid↗
Isolation and characterization of a second molybdopterin dinucleotide: molybdopterin cytosine dinucleotide.
The pterin cofactor (bactopterin) in the molybdoenzyme CO dehydrogenase isolated from Pseudomonas carboxydoflava has previously been shown to differ from molybdopterin in molecular mass, phosphate content, stability, and other properties, implying a novel structure. The structure of the CO dehydrogenase pterin has been investigated in the present studies by alkylation and isolation of the carboxamidomethyl derivative. The alkylated pterin was identified as [di-(carboxamidomethyl)]molybdopterin cytosine dinucleotide on the basis of its absorption properties and by degradation with nucleotide pyrophosphatase yielding carboxamidomethylmolybdopterin and CMP. Further treatment of these products with alkaline phosphatase produced species with absorption and chromatographic properties identical to those of the corresponding dephospho compounds. Molybdopterin cytosine dinucleotide is the second molybdopterin variant to be structurally characterized. The fact that molybdopterin cytosine dinucleotide and molybdopterin guanine dinucleotide contain molybdopterin in their structure shows that the pterin moiety, with its unique dithiolene-containing sidechain, is a structural element which is common to the organic portion of the molybdenum cofactors of many molybdoenzymes.
Aldehyde Oxidoreductases↗
Molybdopterin guanine dinucleotide: a modified form of molybdopterin identified in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitrificans.
The nature of molybdenum cofactor in the bacterial enzyme dimethyl sulfoxide reductase has been investigated by application of alkylation conditions that convert the molybdenum cofactor in chicken liver sulfite oxidase and milk xanthine oxidase to the stable, well-characterized derivative [di(carboxamidomethyl)]molybdopterin. The alkylated pterin obtained from dimethyl sulfoxide reductase was shown to be a modified form of alkylated molybdopterin with increased absorption in the 250-nm region of the spectrum and altered chromatographic behavior. The complex alkylated pterin was resolved into two components by treatment with nucleotide pyrophosphatase. These were identified as di(carboxamidomethyl)molybdopterin and GMP by their absorption spectra, coelution with standard compounds, and by further degradation by alkaline phosphatase to dephospho [di(carboxamidomethyl)]molybdopterin and guanosine. The GMP moiety was sensitive to periodate, identifying it as the 5' isomer. Chemical analysis of the intact alkylated pterin showed the presence of two phosphate residues per pterin. These results established that the pterin isolated from dimethyl sulfoxide reductase contains the phosphoric anhydride of molybdopterin and 5'-GMP, which is designated molybdopterin guanine dinucleotide.
Chromatography, High Pressure Liquid↗
The structure of a molybdopterin precursor. Characterization of a stable, oxidized derivative.
An oxidized pterin species, termed compound Z, has been isolated from molybdenum cofactor-deficient mutants of Escherichia coli and shown to be the direct product of oxidation of a molybdopterin precursor which accumulates in these mutants. The complete structural characterization of compound Z has been accomplished. A carbonyl function at C-1' of the 6-alkyl side chain can be reacted with 2,4-dinitrophenylhydrazine to yield a phenylhydrazone and can be reduced with borohydride, producing a mixture of two enantiomers, each with a hydroxyl group on C-1'. Compound Z contains one phosphate/pterin and no sulfur. The phosphate group is insensitive to alkaline phosphatase and to a number of phosphodiesterases but is quantitatively released as inorganic phosphate by mild acid hydrolysis. From 31P and 1H NMR of compound Z it was inferred that the phosphate is bound to C-2' and C-4' of a 4-carbon side chain, forming a 6-membered cyclic structure. Mass spectral analysis showed an MH+ ion with an exact mass of 344.0401 corresponding to the molecular formula C10H11N5O7P, confirming the proposed structure.
Alkaline Phosphatase↗