Purification and properties of dimethylsulfoxide reductase containing a molybdenum cofactor from a photodenitrifier, Rhodopseudomonas sphaeroides f.s. denitrificans

Dimethylsulfoxide (DMSO) reductase was purified to electrophoretic homogeneity from the periplasmic fraction of a photodenitrifier, Rhodopseudomonas sphaeroides f.s. denitrificans. The enzyme had a molecular weight of 82,000 and had no subunit. It contained 1 mol of molybdenum per mol of enzyme, but iron and acid-labile sulfur were not present. The UV-visible spectrum showed only one absorption maximum at 280 nm. Denaturation of the enzyme released a molybdopterin cofactor, the fluorescence spectra of which were almost the same as those of a form B derivative of molybdopterin found in formate dehydrogenase. The Km value for DMSO was 15 microM, which was much lower than that for trimethylamin-N-oxide (TMAO), whereas Vmax with TMAO was larger than that with DMSO.     

Purification and properties of trimethylamine N-oxide reductase from aerobic photosynthetic bacterium Roseobacter denitrificans.

Trimethylamine N-oxide (TMAO) reductase was purified from an aerobic photosynthetic bacterium Roseobacter denitrificans. The enzyme was purified from cell-free extract by ammonium sulfate fractionation, DEAE ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme was composed of two identical subunits with molecular weight of 90,000, as identified by SDS-polyacrylamide gel electrophoresis, containing heme c and a molybdenum cofactor. The molecular weight of the native enzyme determined by gel filtration was 172,000. The midpoint redox potential of heme c was +200 mV at pH 7.5. Absorption maxima appeared at 418,524, and 554 nm in the reduced state and 410 nm in the oxidized state. The enzyme reduced TMAO, nicotine acid N-oxide, picoline N-oxide, hydroxylamine, and bromate, but not dimethyl sulfoxide, methionine sulfoxide, chlorate, nitrate, or thiosulfate. Cytochrome c2 served as a direct electron donor. It probably catalyzes the electron transfer from cytochrome b-c1 complex to TMAO reductase. Cytochrome c552, another soluble low-molecular-weight cytochrome of this bacterium, also donated electrons directly to TMAO reductase.     

Purification and properties of Escherichia coli dimethyl sulfoxide reductase, an iron-sulfur molybdoenzyme with broad substrate specificity.

Dimethyl sulfoxide reductase, a terminal electron transfer enzyme, was purified from anaerobically grown Escherichia coli harboring a plasmid which codes for dimethyl sulfoxide reductase. The enzyme was purified to greater than 90% homogeneity from cell envelopes by a three-step purification procedure involving extraction with the detergent Triton X-100, chromatofocusing, and DEAE ion-exchange chromatography. The purified enzyme was composed of three subunits with molecular weights of 82,600, 23,600, and 22,700 as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was determined by gel electrophoresis to be 155,000. The purified enzyme contained 7.5 atoms of iron and 0.34 atom of molybdenum per mol of enzyme. The presence of molybdopterin cofactor in dimethyl sulfoxide reductase was identified by reconstitution of cofactor-deficient NADPH nitrate reductase activity from Neurospora crassa nit-I mutant and by UV absorption and fluorescence emission spectra. The enzyme displayed a very broad substrate specificity, reducing various N-oxide and sulfoxide compounds as well as chlorate and hydroxylamine.     

Molybdopterin guanine dinucleotide is the organic moiety of the molybdenum cofactor in respiratory nitrate reductase from Pseudomonas stutzeri

Abstract Respiratory nitrate reductase from the denitrifying bacterium Pseudomonas stutzeri is an iron-sulfur enzyme containing the molybdenum cofactor. Hydrolysis of native nitrate reductase with aqueous sulfuric acid revealed 0.92 mol of 5'-GMP per mol of enzyme. The pterin present in the molybdenum cofactor was liberated from the protein and reacted with iodoacetamide. The resulting di(carboxamidomethyl) (cam) derivative was purified on a C₁₈-cartridge and analyzed for its structural elements. Treatment of the cam derivative with nucleotide pyrophosphatase and subsequent HPLC analysis revealed the formation of di(cam)molybdopterin and 5'-GMP at a 1:1 molar ratio and with a yield of 79% with respect to the molybdenum content of the enzyme. Treatment of the cam derivative with nucleotide pyrophosphatase and alkaline phosphatase led to the liberation of 0.51 mol dephosphodi(cam)molybdopterin and of 0.59 mol guanosine per mol of enzyme, which is equal to a molar ratio of 1:2.2. The results indicate, that the organic moiety of the molybdenum cofactor of nitrate reductase from P. stutzeri is molybdopterin guanine dinucleotide of which one mol is contained per mol of nitrate reductase.     

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.     

Purification and characterization of a molybdenum-pterin-binding protein (Mop) in Clostridium pasteurianum W5.

A large-scale fractionation scheme purified the major molybdenum(Mo)-binding protein (Mop) from crude extracts of Clostridium pasteurianum, with a 10 and 0.2% yield of Mo and protein, respectively. The apparent molecular weight of the purified molybdoprotein is 5,700, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein contains 0.7 mol of Mo per mol of protein with a molecular weight of 5,700. Mop, as isolated, has a peak absorbency at 293 nm. Denaturation and oxidation of the molybdoprotein released multiple pterin like fluorescent compounds. Mop appears to contain a pterin derivative and Mo, but phosphate analysis indicated that the pterin at the very least is not phosphorylated; phosphorylation is required for functional molybdenum cofactor. All treatments used to release the putative Mo-pterin species from Mop failed to yield a molybdopterin that had detectable molybdenum cofactor activity.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Characterisation of the mob locus of Rhodobacter sphaeroides WS8: mobA is the only gene required for molybdopterin guanine dinucleotide synthesis

The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molybdopterin guanine dinucleotide. The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a molybdopterin enzyme, is unaffected. The inability of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.     

Studies on nitrate reductase of Clostridium perfringens. IV. Identification of metals, molybdenum cofactor, and iron-sulfur cluster

Nitrate reductase of Clostridium perfringens was purified by an improved method using immuno-affinity chromatography. The purified preparation contained Mo, Fe, and acid-labile sulfide; the Mo content was 1 mol per mol and the Fe 3.7 mol per mol of the enzyme. The inactive enzyme obtained from cells grown in the presence of tungstate did not hold Mo but contained 1 mol of W. The content of Fe was not increased. The presence of molybdenum cofactor in the nitrate reductase was indicated by the formation of molybdopterin form A in the oxidation of the enzyme by iodine and by the complementation of NADPH-nitrate reductase with the heart-treated enzyme in the extract of Neurospora crassa nit-1. The Clostridium nitrate reductase had an absorption maximum at 279 nm and shoulders at 320, 380, 430, and 520 nm. This enzyme seems to contain an iron sulfur cluster since the reduced enzyme showed decreased absorption in visible region. The CD spectrum of the enzyme has a positive peak at 425 nm and negative ones at 310, 360, and 595 nm. It was compared with the CD spectrum of ferredoxin (2Fe-2S or 4Fe-4S cluster) and the nitrate reductase of Plectonema boryanum.     

Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum

Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)(2) subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.     

Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase.

The molybdopterin cofactor from the formate dehydrogenase of Methanobacterium formicicum was studied. The cofactor was released by guanidine denaturation of homogeneous enzyme, which also released greater than 80% of the molybdenum present in the enzyme. The anoxically isolated cofactor was nonfluorescent, but after exposure to air it fluoresced with spectra similar to those of described molybdopterin cofactors. Aerobic release from acid-denatured formate dehydrogenase in the presence of I2 and potassium iodide produced a mixture of fluorescent products. Alkaline permanganate oxidation of the mixture yielded pterin-6-carboxylic acid as the only detectable fluorescent product. The results showed that the cofactor from formate dehydrogenase contained a pterin nucleus with a 6-alkyl side chain of unknown structure. Covalently bound phosphate was also present. The isolated cofactor was unable to complement the cofactor-deficient nitrate reductase of the Neurospora crassa nit-1 mutant.     

Formation of thieno[3,2-g]pterines from the molybdenum cofactor

A fluorescent oxidation product of the molybdenum cofactor was isolated from Escherichia coli nitrate reductase (EC 1.9.6.1) and bovine milk xanthine oxidase (EC 1.2.3.2), which showed a visible absorption band at 395 nm and was dephosphorylated by alkaline phosphatase but not by phosphodiesterase I. The dephosphorylated species was oxidized by periodate to thieno[3,2-g]pterin-2-carbaldehyde which was quantitatively converted to thieno[3,2-g]pterin-2-carboxylic acid by subsequent treatment with Ag2O in 2 N NaOH. These results indicate that the oxidation product of the molybdenum cofactor is a thieno[3,2-g]pterin derivative with an unidentified side chain in the 2 position.     

Molybdenum cofactor from the cytoplasmic membrane of Proteus mirabilis

Molybdenum cofactor was extracted from membranes of Proteus mirabilis by three methods: acidification, heat treatment and heat treatment in the presence of sodium-dodecylsulphate (SDS). Extracts prepared by the latter method contained the highest concentration of molybdenum cofactor. In these extracts molybdenum cofactor was present in a low molecular weight form. It could not penetrate an YM-2 membrane during ultrafiltration suggesting a molecular weight above 1000. During aerobic incubation of cofactor extracts from membranes at least four fluorescent species were formed as observed in a reversed-phase high performance liquid chromatography (HPLC) system. The species in the first peak was inhomogeneous while the species in the others seem to be homogenous. In water, all fluorescent products had an excitation maximum at 380 nm and an emission maximum at 455 nm. Their absorption spectra showed maxima at around 270 nm and 400 nm. Fluorescent compounds present in the first peak could penetrate an YM-2 membrane during ultrafiltration, whereas the compounds in the other peaks hardly did. Using xanthine oxidase from milk as source of molybdenum cofactor apparently identical cofactor species were found. Cytoplasmic nor membrane extracts of the chlorate resistant mutant chl S 556 of P. mirabilis could complement nitrate reductase of Neurospora crassa nit-1 in the presence of 20 mM molybdate. However, fluorescent species with identical properties as found for the wild-type were formed during aerobic incubation of extracts from membranes of this mutant.Non-common Abbreviations HPLC high performance liquid chromatography - I.D. internal diameter - SDS sodium dodecyl sulphate     

Isolation and characterization of R-primes of Azotobacter vinelandii

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.     

Molybdopterin in carbon monoxide oxidase from carboxydotrophic bacteria.

The carbon monoxide oxidases (COXs) purified from the carboxydotrophic bacteria Pseudomonas carboxydohydrogena and Pseudomonas carboxydoflava were found to be molybdenum hydroxylases, identical in cofactor composition and spectral properties to the recently characterized enzyme from Pseudomonas carboxydovorans (O. Meyer, J. Biol. Chem. 257:1333-1341, 1982). All three enzymes exhibited a cofactor composition of two flavin adenine dinucleotides, two molybdenums, eight irons and eight labile sulfides per dimeric molecule, typical for molybdenum-containing iron-sulfur flavoproteins. The millimolar extinction coefficient of the COXs at 450 nm was 72 (per two flavin adenine dinucleotides), a value similar to that of milk xanthine oxidase and chicken liver xanthine dehydrogenase at 450 nm. That molybdopterin, the novel prosthetic group of the molybdenum cofactor of a variety of molybdoenzymes (J. Johnson and K. V. Rajagopalan, Proc. Natl. Acad. Sci. U.S.A. 79:6856-6860, 1982) is also a constituent of COXs from carboxydotrophic bacteria is indicated by the formation of identical fluorescent cofactor derivatives, by complementation of the nitrate reductase activity in extracts of Neurospora crassa nit-l, and by the presence of organic phosphate additional to flavin adenine dinucleotides. Molybdopterin is tightly but noncovalently bound to the protein. COX, sulfite oxidase, xanthine oxidase, and xanthine dehydrogenase each contains 2 mol of molybdopterin per mol of enzyme. The presence of a trichloroacetic acid-releasable, so-far-unidentified, phosphorous-containing moiety in COX is suggested by the results of phosphate analysis.     

Microbial metabolism of quinoline and related compounds. VII. Quinoline oxidoreductase from Pseudomonas putida: a molybdenum-containing enzyme

The quinoline oxidoreductase from Pseudomonas putida was purified 50-fold to homogeneity with 21% recovery, using ammonium sulfate precipitation, hydrophobic interaction-, anion exchange-, and gel chromatography. The Mr of the native enzyme was calculated to be 300,000 by gel filtration. SDS-polyacrylamide gel electrophoresis of the enzyme revealed three protein bands corresponding to Mr 85,000, 30,000 and 20,000. The enzyme contained 8 atoms of iron, 8 atoms of acid-labile sulfide, 2 molecules of FAD, and the molybdenum cofactor, molybdopterin. Besides quinoline, the quinoline oxidoreductase also catalysed the conversion of 5-, 6-, 7- and 8-hydroxyquinoline and 8-chloroquinoline to the corresponding 2-oxo compounds. The incorporated oxygen atom was derived from water. Cyanide and methanol were effective inhibitors.     

In vitro reconstitution of nitrate reductase activity of the Neurospora crassa mutant nit-1: specific incorporation of molybdopterin

The reduced, metal-free pterin of the molybdenum cofactor has been termed molybdopterin. Oxidation of any molybdopterin-containing protein in the presence or absence of iodine yields oxidized molybdopterin derivatives termed Form A and Form B, respectively. Application of these procedures to whole cells and cell extracts has demonstrated the presence of molybdopterin in wild-type Neurospora crassa, and its absence in the cofactor-deficient mutant nit-1. In order to demonstrate that the reconstitution of nitrate reductase activity in nit-1 extracts results from the incorporation of molybdopterin into the apoprotein, active molybdopterin, free of contaminating amino acids or peptides, was isolated from chicken liver sulfite oxidase and used in the reconstitution system. The results show that, during reconstitution, exogenous molybdopterin is specifically incorporated into the nitrate reductase protein, confirming the role of molybdopterin as the organic moiety of the molybdenum cofactor.     

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.     

Composition of the coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum.

The coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum was purified to electrophoretic homogeneity by anoxic procedures which included the addition of azide, flavin adenine dinucleotide (FAD), glycerol, and 2-mercaptoethanol to all buffer solutions to stabilize activity. The enzyme contains, in approximate molar ratios, 1 FAD molecule and 1 molybdenum, 2 zinc, 21 to 24 iron, and 25 to 29 inorganic sulfur atoms. Denaturation of the enzyme released a molybdopterin cofactor. The enzyme has a molecular weight of 177,000 and consists of one each of two different subunits, giving the composition alpha 1 beta 1. The molecular weight of the alpha-subunit is 85,000, and that of the beta-subunit is 53,000. The UV-visible spectrum is typical of nonheme iron-sulfur flavoprotein. Reduction of the enzyme facilitated dissociation of FAD, and the FAD-depleted enzyme was unable to reduce coenzyme F420. Preincubation of the FAD-depleted enzyme with FAD restored coenzyme F420-dependent activity.     

The molybdenum cofactor of formylmethanofuran dehydrogenase from Methanosarcina barkeri is a molybdopterin guanine dinucleotide

The molybdenum cofactor of formylmethanofuran dehydrogenase from methanol-grown Methanosarcina barkeri was isolated as the [di(carboxamidomethyl)]-derivative. The alkylated factor showed an absorption spectrum and chemical properties identical to those recently reported for the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides. By treatment with nucleotide pyrophosphatase the factor was resolved into two components, which were identified as [di(carboxamidomethyl)]-molybdopterin and GMP by their absorption spectra, their retention times on Lichrospher RP-18, and by their conversion to dephospho-[di(carboxamidomethyl)]-molybdopterin and guanosine, respectively, in the presence of alkaline phosphatase. The GMP-moiety was sensitive to periodate, identifying it as the 5'-isomer. These results demonstrate that the molybdenum cofactor isolated from formylmethanofuran dehydrogenase contains the phosphoric anhydride of molybdopterin and 5'-GMP.