Guinea pig liver aldehyde oxidase as a sulfoxide reductase: Its purification and characterization

Guinea pig aldehyde oxidase was purified about 120-fold at a yield of 26% from liver cytosol by sequential column chromatography using DEAE-cellulose, FMN-Sepharose 4B, and Sephacryl S-300. The purified enzyme showed many similarities with the rabbit liver aldehyde oxidase reported by other workers with respect to its absolute spectra, molecular weight, and cofactor compositions of molybdenum, FAD, and nonheme iron. This enzyme efficiently utilized 2-hydroxypyrimidine and benzaldehyde as electron donors while N¹-methylnicotinamide was 40 times less effective than 2-hydroxypyrimidine. Diphenyl sulfoxide was reduced anaerobically to diphenyl sulfide in the presence of electron donors. This activity was highly susceptible to SKF 525-A as well as the known inhibitors for aldehyde oxidase such as menadione, estradiol, and potassium cyanide. This enzyme also reduced dibenzyl sulfoxide, phenothiazine sulfoxide, d-biotin methyl ester d-sulfoxide, and quinoline N-oxide, but not l-methionine sulfoxide, dimethyl sulfoxide, d-biotin methyl ester l-sulfoxide, and d-biotin d- and l-sulfoxides, as well as diphenyl sulfone. These results indicate that aldehyde oxidase in guinea pig liver functions as a sulfoxide reductase with selective substrate specificity under anaerobic conditions.     

Oxidation of Dimethyl Sulfide to Dimethyl Sulfoxide by Phototrophic Purple Bacteria

Enrichment cultures of phototrophic purple bacteria rapidly oxidized up to 10 mM dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO). DMSO was qualitatively identified by proton nuclear magnetic resonance. By using a biological assay, DMSO was always quantitatively recovered from the culture media. DMS oxidation was not detected in cultures incubated in the dark, and it was slow in cultures exposed to full daylight. Under optimal conditions, the second-order rate constant for DMS oxidation was 6 day⁻¹ mg of protein⁻¹ ml⁻¹. The rate constant was reduced in the presence of high concentration of sulfide (>1 mM), but was not affected by the addition of acetate. DMS was also oxidized to DMSO by a pure strain (tentatively identified as a Thiocystis sp.) isolated from the enrichment cultures. DMS supported growth of the enrichment cultures and of the pure strain by serving as an electron source for photosynthesis. A determination of the amount of protein produced in the cultures and an estimation of the electron balance suggested that the two electrons liberated during the oxidation of DMS to DMSO were quantitatively used to reduce carbon dioxide to biomass. The oxidation of DMS by phototrophic purple bacteria may be an important source of DMSO detected in anaerobic ponds and marshes.     

Utilization of Dimethyl Sulfide as a Sulfur Source with the Aid of Light by Marinobacterium sp. Strain DMS-S1

Strain DMS-S1 isolated from seawater was able to utilize dimethyl sulfide (DMS) as a sulfur source only in the presence of light in a sulfur-lacking medium. Phylogenetic analysis based on 16S ribosomal DNA genes indicated that the strain was closely related to Marinobacterium georgiense. The strain produced dimethyl sulfoxide (DMSO), which was a main metabolite, and small amounts of formate and formaldehyde when grown on DMS as the sole sulfur source. The cells of the strain grown with succinate as a carbon source were able to use methyl mercaptan or methanesulfonate besides DMS but not DMSO or dimethyl sulfone as a sole sulfur source. DMS was transformed to DMSO primarily at wavelengths between 380 and 480 nm by heat-stable photosensitizers released by the strain. DMS was also degraded to formaldehyde in the presence of light by unidentified heat-stable factors released by the strain, and it appeared that strain DMS-S1 used the degradation products, which should be sulfite, sulfate, or methanesulfonate, as sulfur sources.     

Methionine sulfoxide reduction in ciliates: Characterization of the ready-to-use methionine sulfoxide-R-reductase genes in Euplotes

Genes encoding the enzyme methionine sulfoxide reductase type B, specific to the reduction of the oxidized methionine-R form, were characterized from the expressed (macronuclear) genome of two ecologically separate marine species of Euplotes, i.e. temperate water E. raikovi and polar water E. nobilii. Both species were found to contain a single msrB gene with a very simple structural organization encoding a protein of 127 (E. raikovi) or 126 (E. nobilii) amino acid residues that belongs to the group of zinc-containing enzymes. Both msrB genes are constitutively expressed, suggesting that the MsrB enzyme plays an essential role in repairing oxidative damages that appear to be primarily caused by physiological cell aging in E. raikovi and by interactions with an O₂ saturated environment in E. nobilii.     

Inactivation of MXR1 Abolishes Formation of Dimethyl Sulfide from Dimethyl Sulfoxide in Saccharomyces cerevisiae

Dimethyl sulfide (DMS) is a sulfur compound of importance for the organoleptic properties of beer, especially some lager beers. Synthesis of DMS during beer production occurs partly during wort production and partly during fermentation. Methionine sulfoxide reductases are the enzymes responsible for reduction of oxidized cellular methionines. These enzymes have been suggested to be able to reduce dimethyl sulfoxide (DMSO) as well, with DMS as the product. A gene for an enzymatic activity leading to methionine sulfoxide reduction in Saccharomyces yeast was recently identified. We confirmed that the Saccharomyces cerevisiae open reading frame YER042w appears to encode a methionine sulfoxide reductase, and propose the name MXR1 for the gene. We found that Mxr1p catalyzes reduction of DMSO to DMS and that an mxr1 disruption mutant cannot reduce DMSO to DMS. Mutant strains appear to have unchanged fitness under several laboratory conditions, and in this paper I hypothesize that disruption of MXR1 in brewing yeasts would neutralize the contribution of the yeast to the DMS content in beer.     

Oxidation of dimethyl sulfide byPseudomonas acidovorans DMR-11 isolated from peat biofilter

Summary Pseudomonas acidovorans DMR-11, capable of oxidizing dimethyl sulfide (DMS), was isolated from peat biofilter. DMS as a sole carbon or energy source was not degraded, but it was co-degraded in the medium containing organic carbon sources. The removal rate of DMS in heat-treated glucose medium was 1.12×10^–17 mole/h cell at 30 °C. Dimethyl sulfoxide (DMSO) was the only product of DMS oxidation and was formed stoichiometrically. DMS was reversibly evolved in excess of DMSO. The cell free extract of strain DMR-11 oxidized DMS in presence of NADPH.     

Isolation of Rhodococcus sp. Strain ECU0066, a New Sulfide Monooxygenase-Producing Strain for Asymmetric Sulfoxidation

A new and efficient sulfide monooxygenase-producing strain, ECU0066, was isolated and identified as a Rhodococcus sp. that could transform phenylmethyl sulfide (PMS) to (S)-sulfoxide with 99% enantiomeric excess via two steps of enantioselective oxidations. Its enzyme activity could be effectively induced by adding PMS or phenylmethyl sulfoxide (PMSO) directly to a rich medium at the early log phase (6 h) of fermentation, resulting in over 10-times-higher production of the enzyme. This bacterial strain also displayed fairly good activity and enantioselectivity toward seven other sulfides, indicating a good potential for practical application in asymmetric synthesis of chiral sulfoxides.     

A new fungus which degrades hydrogen sulfide,methanethiol, dimethyl sulfide and dimethyl disulfide

Summary A new Basidiomycete showed significantly higher degradation rates, 10,000 times for H₂S,40 times for dimethyl sulfide(DMS),15 times for methanethiol(MT) and 4 times for dimethyl disulfide(DMDS) than any reported previously. The optimal pH for degradation activity was around 7. Degradation rate for each gas when mixed gases of H₂S,MT and DMS were supplied was almost the same as that for single gas supply. H₂S was oxidized to SO₄ via SO₃ and DMS was stoichiometrically converted to dimethyl sulfoxide(DMSO).     

Anaerobic induction of trimethylamine N-oxide reductase and cytochromes by dimethyl sulfoxide inEscherichia coli

Reduction of trimethylamine N-oxide is catalyzed by at least two enzymes inEscherichia coli: trimethylamine N-oxide reductase, which is anaerobically induced by trimethylamine N-oxide, and the constitutive enzyme dimethyl sulfoxide reductase. In this study, an increase in the specific activity of trimethylamine N-oxide reduction was observed in the anaerobic culture with dimethyl sulfoxide, but the specific activity of dimethyl sulfoxide reduction was not changed. The inducible enzyme trimethylamine N-oxide reductase was found in this culture. A marked expression of the structural genetorA for trimethylamine N-oxide reductase was also observed in atorA-lacZ gene fusion strain under anaerobic conditions with either trimethylamine N-oxide or dimethyl sulfoxide.l-Methionine sulfoxide and the N-oxides of adenosine, picolines, and nicotinamide slightly repressed expression of the gene. Membrane-boundb- andc-type cytochromes involved in the trimethylamine N-oxide reduction were also produced in a wild-type strain grown anaerobically with dimethyl sulfoxide. But thec-type cytochrome was not produced in thetorA-lacZ strain grown anaerobically with trimethylamine N-oxide or dimethyl sulfoxide; this suggests that there is a correlation between the expression oftorA and the synthesis of the cytochrome.     

Effects of dimethyl sulfoxide and glycerol on catalytic and regulatory properties of glutamine-dependent carbamoyl phosphate synthase from rat liver and dual effects of uridine triphosphate.

The cryoprotectants dimethyl sulfoxide and glycerol markedly affected the activity of glutamine-dependent carbamoyl phosphate synthase (EC 2.7.2.9) of rat liver, the first enzyme of de novo pyrimidine biosynthesis. The apparent K_m for MgATP^2? decreased with increases in the solvent concentrations, from 7.0 mm without the solvents to 0.1 and 0.8 mm in the presence of 25% ($\text{v}\text{v}$) dimethyl sulfoxide and 30% ($\text{w}\text{w}$) glycerol, respectively. The apparent K_m for bicarbonate also decreased with these solvents, while that for glutamine was not significantly affected. The response in the maximal velocity to the solvents was biphasic; the value of V increased 1.39- and 1.18-fold with 7.5% dimethyl sulfoxide and 10% glycerol, respectively, but then decreased as the concentrations of the solvents were further increased. The extents of inhibition by 25% dimethyl sulfoxide and 30% glycerol were 63 and 44%, respectively. The effects of 5-phosphoribosyl 1-pyrophosphate and MgUTP, allosteric effectors, were greatly modified by these solvents. Kinetic parameters as affected by the effectors in the presence of various concentrations of the solvents are described. A notable observation was that MgUTP, a potent inhibitor under ordinary conditions, stimulated the activity in the presence of high concentrations of dimethyl sulfoxide; in the presence of 30% dimethyl sulfoxide and 1 mm MgATP^2?, 0.5 to 2.0 mm MgUTP enhanced the enzymatic activity about twofold. MgUTP at higher concentrations was inhibitory. The dimethyl sulfoxide-dependent dual effects of MgUTP indicate the possible existence of at least two MgUTP-binding sites on the enzyme molecule.     

Growth of Escherichia coli Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source

Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source. Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled E. coli to grow when dimethyl phosphate is the sole phosphorus source. E. coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled E. coli to use phosphotriesters as a sole source of phosphorus.     

Site-directed mutagenesis of dimethyl sulfoxide reductase from Rhodobacter capsulatus: characterization of a Y114 --> F mutant

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.     

Fermentation and anaerobic respiration by Rhodospirillum rubrum and Rhodopseudomonas capsulata

Rhodospirillum rubrum and Rhodopseudomonas capsulata were able to grow anaerobically in the dark either by a strict mixed-acid fermentation of sugars or, in the presence of an appropriate electron acceptor, by an energy-linked anaerobic respiration. Both species fermented fructose without the addition of accessory oxidants, but required the initial presence of bicarbonate before fermentative growth could begin. Major products of R. rubrum fermentation were succinate, acetate, propionate, formate, hydrogen, and carbon dioxide; R. capsulata produced major amounts of lactate, acetate, succinate, hydrogen, and carbon dioxide. R. rubrum and R. capsulata were also capable of growing strictly through anaerobic, respiratory mechanisms. Nonfermentable substrates, such as succinate, malate, or acetate, supported growth only in the presence of an electron acceptor such as dimethyl sulfoxide or trimethylamine oxide. Carbon dioxide and dimethyl sulfide were produced during growth of R. rubrum and R. capsulata on succinate plus dimethyl sulfoxide. Molar growth yields from cultures grown anaerobically in the dark on fructose plus dimethyl sulfoxide were 3.8 to 4.6 times higher than values obtained from growth on fructose alone and were 56 to 60% of the values obtained from aerobic, respiratory growth with fructose. Likewise, molar growth yields from anaerobic, respiratory growth conditions with succinate plus dimethyl sulfoxide were 51 to 54% of the values obtained from aerobic, respiratory growth with succinate. The data indicate that dimethyl sulfoxide or trimethylamine oxide as a terminal oxidant is approximately 33 to 41% as efficient as O₂ in conserving energy through electron transport-linked respiration.     

Cryoenzymology of β-galactosidase: Effects of cryosolvents

Experimental support for the use of fluid aqueous organic solvent systems and subzero temperatures in mechanistic studies of β-galactosidase is presented. The enzyme was stable and retained catalytic activity and structural integrity in 50% aqueous dimethyl sulfoxide and 60% aqueous methanol at 0°C; at lower temperatures higher concentrations of cosolvent may be successfully used. The effects of dimethyl sulfoxide on the catalytic and structural properties of the enzyme were investigated in detail. For the β-galactoside-catalyzed h ydrolysis ofo-nitrophenyl-β-D-galactoside the value ofk _cat decreased in a linear manner with increasing cosolvent concentration, whereasK _m increased exponentially. The decrease ink _cat paralleled the decrease in water concentration, consistent with rate-limiting hydrolysis of a galactosylenzyme intermediate. The increase inK _m is attributed to less favorable partitioning of the substrate to the active site in the cryosolvent compared to aqueous solution. ThepH*-rate profile for this reaction at 0°C in 50% dimethyl sulfoxide was similar to that in aqueous solution, withpK*₁=5.8 andpK*₂=8.0. Linear Arrhenius plots, with energies of activation of 13.9 and 16.0 kcal mol^?1, respectively, were obtained for the β-galactosidase-catalyzed hydrolysis ofo-nitrophenyl- andp-nitrophenyl-β-D-galactosides in 50% dimethyl sulfoxide at temperatures to ?57°C. Examination of the intrinsic fluorescence and ultraviolet spectra of the enzyme as a function of increasing cosolvent concentration showed no evidence for structural perturbation up to and including 50% dimethyl sulfoxide at 0°C. We conclude that these cryosolvent systems are suitable for mechanistic investigations of β-galactosidase, in particular for trapping intermediates at subzero temperatures.     

Mechanistic studies of Rhodobacter sphaeroides Me2SO reductase

Studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides have yielded new insight into its catalytic mechanism. A series of reductive titrations, performed over the pH range 6-10, reveal that the absorption spectrum of reduced enzyme is highly sensitive to pH. The reaction of reduced enzyme with dimethyl sulfoxide is found to be clearly biphasic throughout the pH range 6-8 with a fast, initial substrate-binding phase and substrate-concentration independent catalytic phase. The intermediate formed at the completion of the fast phase has the characteristic absorption spectrum of the established dimethyl sulfoxide-bound species. Quantitative reductive and oxidative titrations of the enzyme demonstrate that the molybdenum center takes up only two reducing equivalents, implying that the two pyranopterin equivalents of the molybdenum center are not formally redox active. Finally, the visible spectrum associated with the catalytically relevant "high-g split" Mo(V) species has been determined. Spectral deconvolution and EPR quantitation of enzyme-monitored turnover experiments with trimethylamine N-oxide as substrate reveal that no substrate-bound intermediate accumulates and that Mo(V) content remains near unity for the duration of the reaction. Similar experiments with dimethyl sulfoxide show that significant quantities of both the Mo(V) species and the dimethyl sulfoxide-bound complex accumulate during the course of reaction. Accumulation of the substrate-bound complex in the steady-state with dimethyl sulfoxide arises from partial reversal of the physiological reaction in which the accumulating product, dimethyl sulfide, reacts with oxidized enzyme to yield the substrate-bound intermediate, a process that significantly slows turnover.     

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k_cat than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.     

Redox-Dependent Gene Regulation in Rhodobacter sphaeroides 2.4.1T: Effects on Dimethyl Sulfoxide Reductase (dor) Gene Expression

The ability of Rhodobacter sphaeroides 2.4.1^T to respire anaerobically with the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) is manifested by the molybdoenzyme DMSO reductase, which is encoded by genes of the dor locus. Previously, we have demonstrated that dor expression is regulated in response to lowered oxygen tensions and the presence of DMSO or TMAO in the growth medium. Several regulatory proteins have been identified as key players in this regulatory cascade: FnrL, DorS-DorR, and DorX-DorY. To further examine the role of redox potentiation in the regulation of dor expression, we measured DMSO reductase synthesis and β-galactosidase activity from dor::lacZ fusions in strains containing mutations in the redox-active proteins CcoP and RdxB, which have previously been implicated in the generation of a redox signal affecting photosynthesis gene expression. Unlike the wild-type strain, both mutants were able to synthesize DMSO reductase under strictly aerobic conditions, even in the absence of DMSO. When cells were grown photoheterotrophically, dorC::lacZ expression was stimulated by increasing light intensity in the CcoP mutant, whereas it is normally repressed in the wild-type strain under such conditions. Furthermore, the expression of genes encoding the DorS sensor kinase and DorR response regulator proteins was also affected by the ccoP mutation. By using CcoP-DorR and CcoP-DorY double mutants, it was shown that the DorR protein is strictly required for altered dor expression in CcoP mutants. These results further demonstrate a role for redox-generated responses in the expression of genes encoding DMSO reductase in R. sphaeroides and identify the DorS-DorR proteins as a redox-dependent regulatory system controlling dor expression.