甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶羟基化酶组分的纯化和性质

Methylomonassp．GYJ3菌株中经DEAE-SepharoseCL-6B阴离子交换层析和SephacrylS300凝胶层析分离纯化出甲烷加氧酶羟基化酶组分．经HPLC分析,纯度大于90％,分子量为240kD,纯化倍数为3．9,比活为225nmol环氧丙烷每分钟毫克蛋白．SDS-PAGE表明,羟基化酶由三个亚基组成,亚基分子量为56、43、27kD．ICPAES测定羟基化酶的Fe含量为2.1molFe每摩尔蛋白．HPLC法用于甲烷单加氧酶羟基化酶组分的纯化,纯化的羟基化酶组分比活为541nmol（环氧丙烷）每分钟毫克蛋白,是两步LC法纯化的羟基化酶的两倍,Fe含量为3．78molFe每摩尔蛋白．催化性质研究表明羟基化酶能够被化学还原剂还原为还原态羟基化酶,还原态的羟基化酶单独存在时表现出MMO活性,说明它是MMO活性中心,天然态的羟基化酶单独存在时无MMO活性,加入粗酶液中MMO活性明显增加,说明GYJ3菌中MMO是一个复合酶系．     

Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase

Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O₂- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ)₂ complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the α subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.     

Epoxypropane biosynthesis by whole cell suspension of methanol-growth Methylosinus trichosporium IMV 3011

Methanotrophs containing methane monooxygenase (MMO) can catalyze the epoxidation of propene to epoxypropane. Methane cannot support dense biomass growth due to its low aqueous solubility. Low growth rate is important limiting factor for the application of methanotrophs. Methanol can act as growth substrate, but direct addition of methanol is toxic to most methanotrophs. The MMO activity during growth on methanol is also uncertain. In this paper, methanol-adapted Methylosinus trichosporium IMV 3011 was successfully cultivated at high cell densities using methanol as sole carbon source. A biomass density of 1.68 g dry weight cell l^?1 was achieved and cells contained almost 80% of the MMO activity measured for cells grown with methane. It has been found that methanol can also act as the electron-donating substrate to regenerate the NADH and drive epoxypropane synthesis. The effect of methanol supply on the epoxidation capacity of Methylosinus trichosporium IMV3011 was studied in batch reactor. 0.016% methanol concentration was found to give the highest propene epoxidation capacity.     

Evaluation of diphlorethohydroxycarmalol isolated from Ishige okamurae for radical scavenging activity and its protective effect against H2O2-induced cell damage

In this study, the antioxidant activities of 21 species of marine algae were assessed via an ABTS free radical scavenging assay. The Ishige okamurae extract tested herein evidenced profound free radical scavenging activity, compared to that exhibited by other marine algae extracts. Thus, I. okamurae was selected for use in further experiments, and was partitioned with different organic solvents. Profound radical scavenging activity was detected in the ethyl acetate fraction, and the active compound was identified as the carmalol derivative, diphlorethohydroxycarmalol, which evidenced higher levels of activity than that of commercial antioxidants. Moreover, the protective effects of diphlorethohydroxycarmalol against H₂O₂-induced cell damage were evaluated. Intracellular reactive oxygen species (ROS) were overproduced as the result of the addition of H₂O₂, but this ROS generation was reduced significantly after diphlorethohydroxycarmalol treatment; this corresponds to a significant enhancement of cell viability against H₂O₂-induced oxidative damage. The inhibitory effects of diphlorethohydroxycarmalol against cell damage were determined via comet assay and Hoechst staining assay, and diphlorethohydroxycarmalol was found to exert a positive dose-dependent effect. These results clearly indicate that the diphlorethohydroxycarmalol isolated from I. okamurae exerts profound antioxidant effects against H₂O₂-mediated cell damage, and treatment with this compound may be a potential therapeutic modality for the treatment or prevention of several diseases associated with oxidative stress.     

Purification and Characterization of the Soluble Methane Monooxygenase of the Type II Methanotrophic Bacterium Methylocystis sp. Strain WI 14

Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 μM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an α₂β₂γ₂ structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg²⁺ ions (with a total loss of enzyme activity at 0.01 mM Hg²⁺) and Cu²⁺, Zn²⁺, and Ni²⁺ ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).     

On the mechanisms of the reaction of dodecatungstophosphate with alkyl radicals in aqueous solutions

The reduced lacunary polyoxotungstate, [PW₁₁O₃₉]⁸⁻, reacts with the ^.CH₂CH(OH)CH₃ and ^.CH₂C(CH₃)₂OH radicals via a mechanism involving β-hydroxide elimination to yield propene and 2-methyl propene respectively, and [PW₁₁O₃₉]⁷⁻. [PW₁₁O₃₉]⁸⁻ is also oxidized by methyl radicals in a reaction which yields methane as the major product. It is proposed that the reactions proceed via the formation of short lived transients with W-C σ bonds.     

Solvent effect on hemin-catalyzed polymerization of phenols

Phenol and 4-substituted phenols were polymerized by H₂O₂ as an oxidant and hemin as a catalyst in organic/buffer mixed solvents. The chemical structures of the polymers were similar to those synthesized by the catalysis with horseradish peroxidase. Among the organic solvents used, pyridine gave the highest yield of polymer. Also, the manner of addition of H₂O₂ solution affected the polymer yield.     

In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1

Studies of in vitro processing of precursors of the major chlorophyll a/b-binding polypeptides of Chlamydomonas reinhardtii y-1 were undertaken to define the precursor-product relationships. Analysis of translates, prepared from C. reinhardtii poly(A)-rich RNA in a rabbit reticulocyte lysate system, which were incubated with the soluble fraction from C. reinhardtii cells, showed that the 31,500 relative molecular mass (M_r) precursor was converted to the M_r 29,500 thylakoid membrane polypeptide whereas the M_r 30,000 precursor was converted to the M_r 26,000 product. Furthermore, the M_r 31,500 polypeptide, when bound to antibodies, was not processed to the mature polypeptide of M_r 29,500, although the presence of antibodies did not prevent the precursor of M_r 30,000 from being converted to the mature M_r 26,000 polypeptide. The mature fraction of M_r 26,000, was separated into two bands corresponding to polypeptides 16 and 17 in the electrophoretic system of Chua and Bennoun (1975 Proc Natl Acad Sci USA 72: 2175-2179).

Processing activity was present in the soluble fraction obtained from cells grown in the light or in the dark. Therefore, processing of the precursor polypeptides does not appear to be involved in the regulation by light of the accumulation of these polypeptides in thylakoid membranes.

     

Epoxypropane biosynthesis by Methylomonas sp. GYJ3: batch and continuous studies

Methylomonas sp. GYJ3 is a methanotrophic bacterium containing methane monooxygenase (MMO), which catalyses the epoxidation of propene to epoxypropane. In this study, the cell suspension of Methylomonas sp. GYJ3 has been used for epoxypropane biosynthesis from propene. When propene is epoxidized, the product epoxypropane is not further metabolized and accumulates extracellularly. Unfortunately, continuous production of epoxypropane is usually difficult due to exhaustion of reductant and the accumulation of toxic products. Hence, in order to address these problems, batch experiments were performed to explore the possibility of producing epoxypropane by a co-oxidation process. Methane was chosen as the most suitable electron-donating co-substrate since it did not result in molecular toxicity and provided abundant reductant for epoxidation. It was found that the maximum production of epoxypropane occurred in an atmosphere of 30% methane. Batch experiments also indicated that continuous removal of product was necessary to overcome the inhibition of epoxypropane. In continuous experiments, optimum mixed gaseous substrates were continuously circulated through the stirred tank bioreactor to remove product from the cell suspension. Initial epoxypropane productivity was 268 mol/day. The bioreactor has been allowed to operate continuously for 12 days without obvious loss of epoxypropane productivity, and more than 96% of initial MMO activity was retained.     

Mixed Pollutant Degradation by Methylosinus trichosporium OB3b Expressing either Soluble or Particulate Methane Monooxygenase: Can the Tortoise Beat the Hare?

Methanotrophs have been widely investigated for in situ bioremediation due to their ubiquity and their ability to degrade halogenated hydrocarbons through the activity of methane monooxygenase (MMO). It has been speculated that cells expressing the soluble form of MMO (sMMO) are more efficient in cleaning up sites polluted with halogenated hydrocarbons due to its broader substrate range and relatively fast degradation rates compared cells expressing the other form of MMO, the particulate MMO (pMMO). To examine this issue, the biodegradation of mixtures of chlorinated solvents, i.e., trichloroethylene (TCE), trans-dichloroethylene (t-DCE), and vinyl chloride (VC), by Methylosinus trichosporium OB3b in the presence of methane using either form of MMO was investigated over longer time frames than those commonly used, i.e., days instead of hours. Growth of M. trichosporium OB3b along with pollutant degradation were monitored and analyzed using a simple comparative model developed from the Ω model created for analysis of the competitive binding of oxygen and carbon dioxide by ribulose bisphosphate carboxylase. From these findings, it appears that at concentrations of VC, t-DCE, and TCE greater than 10 μM each, methanotrophs expressing pMMO have a competitive advantage over cells expressing sMMO due to higher growth rates. Despite such an apparent growth advantage, pMMO-expressing cells degraded less of these substrates at these concentrations than sMMO-expressing cells during active growth. If the concentrations were increased to 100 μM, however, not only did pMMO-expressing cells grow faster, they degraded more of these pollutants and did so in a shorter amount of time. These findings suggest that the relative rates of growth substrate and pollutant degradation are important factors in determining which form of MMO should be considered for pollutant degradation.     

The methane monooxygenase gene cluster of Methylosinus trichosporium: cloning and sequencing of the mmoC gene

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. The soluble MMO enzyme complex from Methylosinus trichosporium also oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study we have used heterologous DNA probes from the type X methanotroph Methylococcus capsulatus (Bath) to isolate mmo genes from the type II methanotroph M. trichosporium. We report here that the gene encoding the reductase component, Protein C of MMO, lies adjacent to the genes encoding the other components of soluble MMO in M. trichosporium but is separated by an open reading frame of unknown function, orfY. The complete nucleotide sequence of these genes is presented. Sequence analysis of mmoC indicates that the N-terminus of Protein C has significant homology with 2Fe2S ferredoxins from a wide range of organisms.Abbreviations MMO methane monooxygenase     

Complexome analysis of the nitrite-dependent methanotroph Methylomirabilis lanthanidiphila

The atmospheric concentration of the potent greenhouse gases methane and nitrous oxide (N₂O) has increased drastically during the last century. Methylomirabilis bacteria can play an important role in controlling the emission of these two gases from natural ecosystems, by oxidizing methane to CO₂ and reducing nitrite to N₂ without producing N₂O. These bacteria have an anaerobic metabolism, but are proposed to possess an oxygen-dependent pathway for methane activation. Methylomirabilis bacteria reduce nitrite to NO, and are proposed to dismutate NO into O₂ and N₂ by a putative NO dismutase (NO-D). The O₂ produced in the cell can then be used to activate methane by a particulate methane monooxygenase. So far, the metabolic model of Methylomirabilis bacteria was based mainly on (meta)genomics and physiological experiments. Here we applied a complexome profiling approach to determine which of the proposed enzymes are actually expressed in Methylomirabilis lanthanidiphila. To validate the proposed metabolic model, we focused on enzymes involved in respiration, as well as nitrogen and carbon transformation. All complexes suggested to be involved in nitrite-dependent methane oxidation, were identified in M. lanthanidiphila, including the putative NO-D. Furthermore, several complexes involved in nitrate reduction/nitrite oxidation and NO reduction were detected, which likely play a role in detoxification and redox homeostasis. In conclusion, complexome profiling validated the expression and composition of enzymes hypothesized to be involved in the energy, methane and nitrogen metabolism of M. lanthanidiphila, thereby further corroborating their unique metabolism involved in the environmentally relevant process of nitrite-dependent methane oxidation.     

Inhibition and Labeling of the Plant Plasma Membrane H-ATPase with N-Ethylmaleimide

H⁺-ATPase activity in plasma membranes isolated from Avena sativa root cells is inhibited by N-ethylmaleimide, a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced by ADP, MgADP, and MgATP, but even at 40 millimolar ADP the enzyme is only partially protected against inactivation. When plasma membranes are treated wth N-[2-³H]ethylmaleimide and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, prominent radioactive bands appear at M_r=100,000 and several other positions. However, only radioactivity in the M_r=100,000 protein is reduced by the presence of MgADP. These results provide independent evidence that the M_r=100,000 polypeptide which is observed in purified preparations of the enzyme is the catalytic subunit of the H⁺-ATPase. When tryptic peptides are produced from N-[2-³H]ethylmaleimide labeled M_r=100,000 protein and separated by reverse phase high performance liquid chromatography, two radioactive peaks are observed for which N-[2-³H]ethylmaleimide incorporation is reduced in the presence of MgADP.     

Noninvasive estimation of human tissue respiration with wavelet-analysis of oxygen saturation and blood flow oscillations in skin microvessels

Laser Doppler flowmetry, laser spectrophotometry of oxygen saturation, and the fluorescence determination of the NADH/FAD ratio were carried out in 30 subjects in the upper limb skin zones with and without arteriolovenular anastomoses (AVAs). It was demonstrated that the wavelet-analysis of oxygen saturation and blood flow oscillations in microvessels was an efficient approach to noninvasive estimation of the skin oxygen extraction (OE) and oxygen consumption (OC) rates. OE = (SaO₂ ? SvO₂)/SaO₂, where SaO₂ (%) and SvO₂ (%) are the oxygen saturations of arterial and venular blood, respectively. If the cardiac (A_c, perfusion units, p.u.) to respiratory rhythm amplitude (A_r, p.u.) ratio A_c/A_r ?? 1, SvO₂ = SO₂. If A_c/A_r > 1, SvO₂ = SO₂/(A_c/A_r). OC = M _nutr (SaO₂ ?? SvO₂) in p.u. · %O₂, where M _nutr is the nutritive blood flow value in p.u. M _nutr = M/SI, where SI is the shunting index of blood flow in microvessels. The perfusion, OE, and OC values were higher in the skin with AVAs than in the skin without AVAs. The perfusion and oxygen saturation values were more variable in the skin with AVAs. The oxygen diffusing from the tiniest arterioles and capillaries is the most important for tissue metabolism. The contribution of the total perfusion and the oxygen diffusion from arterioles to tissue metabolism increased under the tissue ischemia conditions.     

Effect of organic solvents on a chloroperoxidase biotransformation

The effect of organic solvents on the chlorination activity of chloroperoxidase (CPO) was identified for use in biotransformations with CPO. CPO was found to chlorinate monochlorodimedon (MCD) in the presence of organic solvents with log P values less than 0. The relative rates of chlorination with chloride ion in the presence of H₂O₂, buffer and 2.5–20% of either dimethyl sulfoxide, N,N-dimethyl formamide, methanol or acetonitrile, were in the range of 10–58% of that in buffer (pH 2.8) at the same reactant concentrations. The presence of such organic solvents was found to alter CPO catalysis by altering the protein conformation and the local environment at the active site. CPO did not display chlorination activity in the presence of organic solvents which had log P values greater than 0.     

Intact form of myeloperoxidase from normal human neutrophils

Myeloperoxidase (donor: H₂O₂ oxidoreductase, EC 1.11.1.7) of human polymorphonuclear neutrophils was purified rapidly in the presence of the protease inhibitors phenylmethanesulfonyl fluoride and pepstatin A. The purified enzyme behaved as a single molecular species in several nondenaturing electrophoretic and chromatographic systems. Peroxidase activity in fresh extracts of neutrophils from 20 normal persons and from 5 patients with polycythemia was electrophoretically identical to purified enzyme. Treatment with trypsin converted myeloperoxidase to multiple electrophoretic forms of active enzyme. Size (M_r ca. 15,000 and ca. 55,000) and stoichiometry of the subunits of purified enzyme, and enzyme M_r ca. 140,000, were compatible with intact myeloperoxidase having an α₂β₂ structure. We found no evidence for electrophoretically detectable genetic polymorphism of myeloperoxidase. Proteolytic degradation of myeloperoxidase probably accounted for electrophoretic heterogeneity of enzyme and for some constituent peptides described previously.     

A Secondary Processing Site in the Precursor of the Small Subunit of Ribulose Bisphosphate Carboxylase of Chlamydomonas reinhardtii y-1

When the precursor of ribulose bisphosphate carboxylase of Chlamydomonas reinhardtii y-1 is bound to antibodies and treated with the soluble cell fraction, it is cleaved to the mature form (M_r 16,500) via an intermediate of M_r 18,500. Although this intermediate has only been observed in vitro, it may be produced during processing of the precursor in vivo.     

Structure-activity relationships of chlorinated benzenes as inducers of different forms of cytochrome P-450 in rat liver

Hexachlorobenzene (HCB) produced increases in ethoxyresorufin (ERR) O-deethylase, aryl hydrocarbon hydroxylase (AHH) and aminopyrine N-demethylase activities in rat liver microsomes which were intermediate between those produced by phenobarbital and 3,4-benzpyrene (BP). α-Naphthoflavone (ANF) selectively inhibited ERR activity in BP and HCB-induced microsomes (94% and 88%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of liver microsomes indicated that HCB did not produce a detectable increase in a polypeptide with electrophoretic properties similar to those of purified cytochrome P-448 (M_r = 56 000). However, HCB did induce a polypeptide with M_r = 53 000 corresponding to one of two polypeptide bands induced by BP. This polypeptide may represent a second form of cytochrome P-448. Purification of HCB to remove possible dibenzo-p-dioxin impurities did not alter the ‘mixed-type’ induction produced by HCB. In contrast to HCB, all other chlorinated benzenes tested resembled phenobarbital as inducers.     

Purification and characterization of the berberine bridge enzyme from berberis beaniana cell cultures

The berberine bridge-forming enzyme (BBE) has been found in 66 samples taken from differentiated plants and from cell suspension cultures. It was purified 450-fold from Berberis beaniana cell cultures by gel-filtration, DEAE and phenyl-Sepharose chromatography, electrophoresis and isoclectric focusing. The enzyme was shown to be homogeneous by gel electrophoresis (M_r = 52 kD ± 4). The enzyme, which requires the presence of oxygen, catalyses the conversion of the (S)-enantiomers of reticuline, protosinomenine and laudanosoline to the corresponding (S)-tetrahydroprotoberberines and released stoichiometric amounts of H₂O₂. Within the cells the enzyme is located in a particle with the density p = 1.14 g/ml.     

Purification and molecular properties of 3 polypeptides released from a highly active O2-evolving photosystem-II preparation by Tris-treatment

Tris-treatment of a highly active O₂-evolving photosystem-II preparation induced release of 3 polypeptides (M_r 33 000, 24 000 and 18 000), concomitant with inhibition of O₂ evolution [FEBS Lett. (1981)_133. 265-268]. The 3 polypeptides were purified with the use of electrofocusing. Isoelectric points of the proteins were 5.1, 6.5 and 9.2 in order of decreasing M_r value. Only a trace amount of histidine, cystein and methionine were detected in these proteins. Based on the amino acid compositions, polarity indexes of the proteins were calculated to be 47–49%, suggesting the 3 proteins to be hydrophilic.