Purification and properties of membrane-bound methane hydroxylase from <Emphasis Type="Italic">Methylococcus capsulatus</Emphasis> (Strain M)

Membrane fraction of Methylococcus capsulatus (strain M) were treated with [¹⁴C]acetylene, an affinity label binding to the active center of membrane-bound methane monooxygenase (MMO). High-purity particulate form of methane hydroxylase (pMH) was obtained by ion exchange and hydrophobic chromatography. According to SDS-PAGE data, the enzyme contained three polypeptides with molecular weights of 47 (α), 27 (β), and 25 (γ) kDa in the ratio 1: 1: 1. The radiolabel was contained in the β-subunit of pMH. The protein contained 1 or 2 atoms of nonheme iron and 2–4 atoms of copper per a minimum molecular weight of 99 kDa. This protein did not oxidize methane or propylene in the presence of NADH but was able to oxidize low quantities of methane in the presence of duroquinol. It was established that methanol dehydrogenase (MD) and NADH oxidoreductase (NADH-OR) are peripheral membrane proteins. Possible causes of low activity of high-purity methane hydroxylase are discussed.     

Characterization and structural analysis of an active particulate methane monooxygenase trimer from Methylococcus capsulatus (Bath)

The oxidation of methane to methanol in methanotrophs is catalyzed by the enzyme methane monooxygenase (MMO). Two distinct forms of this enzyme exist, a soluble cytoplasmic MMO (sMMO) and a membrane-bound particulate form (pMMO). We describe here the biochemical characterization of a stable and active purified pMMO hydroxylase (pMMO-H) and report a three-dimensional (3D) structure, determined by electron microscopy and single-particle analysis at 23 A resolution. Both biochemical and structural data indicate that pMMO hydroxylase is trimeric, with each monomer unit comprised of three polypeptides of 47, 26, and 23 kDa. Comparison of the recent crystal structure [Lieberman, R. L., and Rosenzweig, A. C. (2005) Nature 434, 177] of an uncharacterized pMMO-H complex with the three-dimensional (3D) structure determined here yielded a good match between the principal features and the organization of the enzyme monomers into trimers. The data presented here advance our current understanding of particulate methane monooxygenase function by the characterization of an active form of the enzyme and the corresponding 3D structure.     

The viable but non-culturable state in the human pathogen Vibrio vulnificus

Abstract Genes encoding paniculate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides ( pmoA and amoA ) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (α- and γ-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (β-and y-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the γ-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.     

Physiology, biochemistry, and specific inhibitors of CH4, NH4+, and CO oxidation by methanotrophs and nitrifiers.

Ammonia oxidizers (family Nitrobacteraceae) and methanotrophs (family Methylococcaceae) oxidize CO and CH4 to CO2 and NH4+ to NO2-. However, the relative contributions of the two groups of organisms to the metabolism of CO, CH4, and NH4+ in various environments are not known. In the ammonia oxidizers, ammonia monooxygenase, the enzyme responsible for the conversion of NH4+ to NH2OH, also catalyzes the oxidation of CH4 to CH3OH. Ammonia monooxygenase also mediates the transformation of CH3OH to CO2 and cell carbon, but the pathway by which this is done is not known. At least one species of ammonia oxidizer, Nitrosococcus oceanus, exhibits a Km for CH4 oxidation similar to that of methanotrophs. However, the highest rate of CH4 oxidation recorded in an ammonia oxidizer is still five times lower than rates in methanotrophs, and ammonia oxidizers are apparently unable to grow on CH4. Methanotrophs oxidize NH4+ to NH2OH via methane monooxygenase and NH4+ to NH2OH via methane monooxygenase and NH2OH to NO2- via an NH2OH oxidase which may resemble the enzyme found in ammonia oxidizers. Maximum rates of NH4+ oxidation are considerably lower than in ammonia oxidizers, and the affinity for NH4+ is generally lower than in ammonia oxidizers. NH4+ does not apparently support growth in methanotrophs. Both ammonia monooxygenase and methane monooxygenase oxidize CO to CO2, but CO cannot support growth in either ammonia oxidizers or methanotrophs. These organisms have affinities for CO which are comparable to those for their growth substrates and often higher than those in carboxydobacteria. The methane monooxygenases of methanotrophs exist in two forms: a soluble form and a particulate form. The soluble form is well characterized and appears unrelated to the particulate. Ammonia monooxygenase and the particulate methane monooxygenase share a number of similarities. Both enzymes contain copper and are membrane bound. They oxidize a variety of inorganic and organic compounds, and their inhibitor profiles are similar. Inhibitors thought to be specific to ammonia oxidizers have been used in environmental studies of nitrification. However, almost all of the numerous compounds found to inhibit ammonia oxidizers also inhibit methanotrophs, and most of the inhibitors act upon the monooxygenases. Many probably exert their effect by chelating copper, which is essential to the proper functioning of some monooxygenases. The lack of inhibitors specific for one or the other of the two groups of bacteria hampers the determination of their relative roles in nature.     

The particulate methane monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs

The particulate methane monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate methane monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.     

Towards a unified mechanism of biological methane oxidation

Abstract The biological oxidation of methane to methanol is catalysed by soluble and particulate forms of the enzyme methane monooxygenase. Little information is available regarding the structure and mechanism of the particulate enzyme whereas much is known about the soluble form of the enzyme. This review concentrates on current knowledge of the structure of the components of the soluble methane monooxygenase and draws together these results with those on the kinetics and substrate specificity of the enzyme in a possible chemical mechanism for enzymatic methane oxidation.     

Trypsin-like enzyme activity of the extracellular membrane vesicles of Bacteroides gingivalis W50

Trypsin-like enzyme activity in spent culture media from 3-d-old batch cultures of Bacteroides gingivalis W50 was measured by using the hydrolysis of N alpha-benzoyl-L-arginine-p-nitroanilide. The cell-free culture medium was fractionated by differential centrifugation at 10,000 g and 75,000 g, yielding two particulate fractions and a soluble supernatant fraction. About 80% of the total recoverable activity was associated with the particulate fractions, the remainder being in the supernatant. Electron microscopy of ruthenium-red/osmium stained ultrathin sections of the pellet fractions showed them to be composed of vesicular particles (extracellular vesicles), between 50 and 250 nm in diameter. Enzyme activity in all three fractions was enhanced by dithiothreitol. Gel-permeation chromatography of the soluble fraction yielded one peak of activity which contained 64 kDa and 58 kDa polypeptides. Enzyme activity from the vesicular fractions could be solubilized by sonication, giving a similar chromatographic profile to the supernatant fraction. The main peak of activity was composed of 64 kDa and 58 kDa polypeptides. In addition, there was a higher molecular mass enzyme activity peak composed of the 64 kDa and 58 kDa components along with 111 kDa, 93 kDa and 70 kDa polypeptides. We conclude that the trypsin-like enzyme of B. gingivalis is released as a soluble protein and is also associated with extracellular vesicles, in which it may exist as a soluble component and also as a protein complex.     

Particulate methane monooxygenase genes in methanotrophs.

A 45-kDa membrane polypeptide that is associated with activity of the particulate methane monooxygenase (pMMO) has been purified from three methanotrophic bacteria, and the N-terminal amino acid sequence was found to be identical in 17 of 20 positions for all three polypeptides and identical in 14 of 20 positions for the N terminus of AmoB, the 43-kDa subunit of ammonia monooxygenase. DNA from a variety of methanotrophs was screened with two probes, an oligonucleotide designed from the N-terminal sequence of the 45-kDa polypeptide from Methylococcus capsulatus Bath and an internal fragment of amoA, which encodes the 27-kDa subunit of ammonia monooxygenase. In most cases, two hybridizing fragments were identified with each probe. Three overlapping DNA fragments containing one of the copies of the gene encoding the 45-kDa pMMO polypeptide (pmoB) were cloned from Methylococcus capsulatus Bath. A 2.1-kb region was sequenced and found to contain both pmoB and a second gene, pmoA. The predicted amino acid sequences of these genes revealed high identity with those of the gene products of amoB and amoA, respectively. Further hybridization experiments with DNA from Methylococcus capsulatus Bath and Methylobacter albus BG8 confirmed the presence of two copies of pmoB in both strains. These results suggest that the 45- and 27-kDa pMMO-associated polypeptides of methanotrophs are subunits of the pMMO and are present in duplicate gene copies in methanotrophs.     

甲烷单加氧酶的研究进展

甲烷氧化细菌在转化甲烷制造新型燃料、单细胞蛋白和新功能酶生产、污水处理等方面有着潜在的应用前景,因此,甲烷单加氧酶作为其代谢过程中重要的酶系也受到人们的广泛关注。我们简要综述了近年来对甲烷单加氧酶的性质、结构、催化机理等方面的研究,特别是对颗粒性甲皖单加氧酶的相关性质进行了详细的阐述。     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

Methane monooxygenase mutants of Methylosinus trichosporium constructed by marker-exchange mutagenesis

Abstract Methylosinus trichosporium OB3b synthesizes a soluble cytoplasmic methane monooxygenase when grown in copper-depleted medium and a membrane-bound particulate methane monooxygenase under copper-replete conditions. The genes encoding the hydroxylase component of soluble methane monooxygenase, carried on a plasmid in Escherichia coli , were insertionally inactivated using a kanamycin cassette and transferred back into M. trichosporium by conjugation. Marker-exchange mutagenesis, via a double homologous recombination event, yielded a soluble methane monooxygenase-negative mutant which grew only on methane using the particulate methane monooxygenase during copper-replete growth conditions, thus proving that the two methane oxidation systems in this methanotroph are genetically distinct.     

Aerobic biodegradation of N-nitrosodimethylamine (NDMA) by axenic bacterial strains

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen whose appearance in the environment is related to the release of rocket fuel and to chlorine-based disinfection of water and wastewater. Although this compound has been shown to be biodegradable, there is minimal information about the organisms capable of this degradation, and little is understood of the mechanisms or biochemistry involved. This study shows that bacteria expressing monooxygenase enzymes functionally similar to those demonstrated to degrade NDMA in eukaryotes have the capability to degrade NDMA. Specifically, induction of the soluble methane monooxygenase (sMMO) expressed by Methylosinus trichosporium OB3b, the propane monooxygenase (PMO) enzyme of Mycobacterium vaccae JOB-5, and the toluene 4-monooxygenases found in Ralstonia pickettii PKO1 and Pseudomonas mendocina KR1 resulted in NDMA degradation by these strains. In each of these cases, brief exposure to acetylene gas, a suicide substrate for certain monooxygenases, inhibited the degradation of NDMA. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene monooxygenases found in strains PKO1 and KR1 mimicked the behavior of the parent strains. In contrast, M. trichosporium OB3b expressing the particulate form of MMO, Burkholderia cepacia G4 expressing the toluene 2-monooxygenase, and Pseudomonas putida mt-2 expressing the toluene sidechain monooxygenase were not capable of NDMA degradation. In addition, bacteria expressing aromatic dioxygenases were not capable of NDMA degradation. Finally, Rhodococcus sp. RR1 exhibited the ability to degrade NDMA by an unidentified, constitutively expressed enzyme that, unlike the confirmed monooxygenases, was not inhibited by acetylene exposure.     

Insulin and lipolytic hormones stimulate the same phosphodiesterase isoform in rat adipose tissue

Insulin sensitive phosphodiesterase from rat adipocytes is found in particulate fractions. Solubilisation of the enzyme with triton X-100 yields a preparation containing more than one phosphodiesterase activity as judged by its rate of thermal denaturation at 45 degrees C and by its non-linear kinetic plots. Immunoprecipitation of solubilised activity with a polyclonal antiserum raised against purified insulin-sensitive rat liver phosphodiesterase selected a form of the enzyme which showed a single exponential decay of enzyme activity when heated at 45 degrees C and linear low Km kinetics. Treatment of adipocytes with insulin ACTH, glucagon or isoproterenol stimulated the low Km particulate phosphodiesterase. The hormonal activation was retained following solubilisation and was also seen when activity was immunoprecipitated. It is suggested that all four hormones activate the same form of phosphodiesterase.     

Purification and properties of D-myo-inositol 1,4,5-trisphosphate 3-kinase from rat brain. Susceptibility to calpain

A new, rapid method for purification of inositol(1,4,5)P3 3-kinase in high yield from rat brain is described. Purified enzyme exhibited a polypeptide of Mr = 53,000 on sodium dodecyl sulfate-polyacrylamide gel and a specific activity of 29 mumol/min/mg at 37 degrees C in the absence of calmodulin. Inclusion of calpain inhibitors was critical for obtaining the 53-kDa protein as the major product and 0.1% of the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylamino]-2-propanesulfonate, was necessary to stabilize enzyme activity. In the absence of calpain inhibitors, the 53-kDa protein degraded progressively during purification and yielded a mixture containing polypeptides of various sizes. Relative intensity of these degradation products on sodium dodecyl sulfate-polyacrylamide gel varied from one preparation to another. However, broad band(s) at the 42-45 kDa region and a band at 35 kDa were always weak, while bands of 53, 51, 40 (sometimes doublets), 33, and 32 KDa were usually strong. The fact that all of these polypeptides including the weak bands of 42-45 and 35 kDa were derived from the 53 kDa form was confirmed by their immunocross-reactivity with monoclonal antibodies to the 53 kDa form. When the 51, 40, and a mixture of the 33 and 32 kDa forms were obtained separately and nearly free from other forms, each of them exhibited catalytic activity. Nevertheless, calmodulin binds to polypeptides larger than 35,000 but not to the 33 and 32 kDa forms. Incubation of the purified 53 kDa form with calpain generated a fragmentation pattern nearly identical to that generated during purification in the absence of calpain inhibitors. Incubation with five other endoproteases produced proteolytic fragments slightly different from those by calpain. However, the general fragmentation patterns generated by the proteases were similar, suggesting that inositol(1,4,5)P3 3-kinase contains several motifs susceptible to a variety of proteases.     

Trichloroethylene oxidation by the membrane-associated methane monooxygenase in type I,type II and type X methanotrophs

Trichloroethylene (TCE) oxidation was examined in 9 different methanotrophs grown under conditions favoring expression of the membrane associated methane monooxygenase. Depending on the strain, TCE oxidation rates varied from 1 to 677 pmol/min/mg cell protein. Levels of TCE in the reaction mixture were reduced to below 40 nmolar in some strains. Cells incubated in the presence of acetylene, a selective methane monooxygenase inhibitor, did not oxidize TCE.Cultures actively oxidizing TCE were monitored for the presence of the soluble methane monooxygenase (sMMO) and membrane associated enzyme (pMMO). Transmission electron micrographs revealed the cultures always contained the internal membrane systems characteristic of cells expressing the pMMO. Naphthalene oxidation by whole cells, or by the cell free, soluble or membrane fractions was never observed. SDS denaturing gels of the membrane fraction showed the polypeptides associated with the pMMO. Cells exposed to ¹⁴C-acetylene showed one labeled band at 26 kDa, and this protein was observed in the membrane fraction. In the one strain examined by EPR spectroscopy, the membrane fraction of TCE oxidizing cells showed the copper complexes characteristic of the pMMO. Lastly, most of the strains tested showed no hybridization to sMMO gene probes. These findings show that the pMMO is capable of TCE oxidation; although the rates are lower than those observed for the sMMO.     

>Decolourisation and depolymerisation of solubilised low-rank coal by the white-rot basidiomycete Phanerochaete chrysosporium

Peroxidases secreted by the white-rot basidiomycete Phanerochaete chrysosporium can oxidise a wide range of recalcitrant compounds including lignin and aromatic xenobiotics. Since low-rank coals such as brown coal and lignite retain structural features of the parent lignin, we investigated the possibility that P. chrysosporium is capable of acting on a brown coal, with the production of useful low-molecular-mass compounds. In nitrogen-limiting liquid medium containing 0.03% solubilised Morwell brown coal, P. chrysosporium was found to convert about 85% of the coal after 16 days incubation to a form not recoverable by alkali-washing and acid-precipitation. The modal molecular mass of the residual coal macromolecules was reduced from the initial 65kDa to 32 kDa. Extensive bleaching of the coal coincided with the presence of extracellular lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP), although both LiP and MnP activity were lower in cultures containing coal. These reductions are accounted for by interference with the enzyme assays by solubilised coal and by binding of MnP to precipitated coal. LiP was about eight times more sensitive than MnP to inhibition by solubilised coal. In nitrogen-sufficient medium containing solubilised coal, neither coal modification nor LiP activity were observed, suggesting that LiP is an essential component of the bleaching process.     

Aliphatic and chlorinated alkenes and epoxides as inducers of alkene monooxygenase and epoxidase activities in Xanthobacter strain Py2.

The inducible nature of the alkene oxidation system of Xanthobacter strain Py2 has been investigated. Cultures grown with glucose as the carbon source did not contain detectable levels of alkene monooxygenase or epoxidase, two key enzymes of alkene and epoxide metabolism. Upon addition of propylene to glucose-grown cultures, alkene monooxygenase and epoxidase activities increased and after an 11-h induction period reached levels of specific activity comparable to those in propylene-grown cells. Addition of chloramphenicol or rifampin prevented the increase in the enzyme activities. Comparison of the banding patterns of proteins present in cell extracts revealed that polypeptides with molecular masses of 43, 53, and 57 kDa accumulate in propylene-grown but not glucose-grown cells. Pulse-labeling of glucose-grown cells with [35S]methionine and [35S]cysteine revealed that the 43-, 53-, and 57-kDa proteins, as well as two additional polypeptides with molecular masses of 12 and 21 kDa, were newly synthesized upon exposure of cells to propylene or propylene oxide. The addition to glucose-grown cells of a variety of other aliphatic and chlorinated alkenes and epoxides, including ethylene, vinyl chloride (1-chloroethylene), cis- and trans-1,2-dichloroethylene, 1-chloropropylene, 1,3-dichloropropylene, 1-butylene, trans-2-butylene, isobutylene, ethylene oxide, epichlorohydrin (3-chloro-1,2-epoxypropane), 1,2-epoxybutane, cis- and trans-2,3-epoxybutane, and isobutylene oxide stimulated the synthesis of the five propylene-inducible polypeptides as well as increases in alkene monooxygenase and epoxidase activities. In contrast, acetylene, and a range of aliphatic and chlorinated alkanes, did not stimulate the synthesis of the propylene-inducible polypeptides or the increase in alkene monooxygenase and epoxidase activities.     

Molecular biology and regulation of methane monooxygenase

Methanotrophs are ubiquitous in the environment and play an important role in mitigating global warming due to methane. They are also potentially interesting for industrial applications such as production of bulk chemicals or bioremediation. The first step in the oxidation of methane is the conversion to methanol by methane monooxygenase, the key enzyme, which exists in two forms: the cytoplasmic, soluble methane monooxygenase (sMMO) and the membrane-bound, particulate methane monooxygenase (pMMO). This paper reviews the biochemistry and molecular biology of both forms of MMO. In the past few years there have been many exciting new findings. sMMO components have been expressed in heterologous and homologous hosts. The pMMO has been purified and biochemically studied in some detail and the genes encoding the pMMO have been sequenced. Copper ions have been shown to play a key role in regulating the expression of both MMO enzyme complexes. We also present a model for copper regulation based on results from Northern analysis, primer-extensions and new sequence data, and raise a number of unanswered questions for future studies.