Protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath). A novel regulatory protein of enzyme activity

An understanding of the mechanism of biological methane oxidation has been hampered by the lack of purified proteins. We describe here a purification protocol for the previously uncharacterized protein B of the soluble methane monooxygenase from the obligate methanotroph Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme consisting of a hydroxylase component, protein A, a reductase component, protein C, and protein B. All three proteins are required for monooxygenase activity. Protein B proves to be a low molecular weight (16,000) single subunit protein devoid of prosthetic groups. The protein is a powerful regulator of soluble methane monooxygenase activity, possessing the capacity to convert the enzyme from an oxidase to an oxygenase. Proteins A and C together catalyze the reduction of molecular oxygen to water, a reaction prevented by protein B. The uncoupling of soluble methane monooxygenase in this manner displays a number of novel features. First, the product of the uncoupled reaction is water, and second, the uncoupling is independent of substrate. Free hydrogen peroxide is not an intermediate in the reduction of oxygen by the incomplete methane monooxygenase enzyme complex. Finally, electron transfer can occur between protein C and protein A in the absence of protein B and protein B prevents the steady-state transfer of electrons in the absence of an oxidizable substrate, such as methane. It is demonstrated that oxygen reduction occurs at the active site of the hydroxylase component, protein A. A unifying mechanism, describing the interaction of the three proteins of soluble methane monooxygenase, is proposed.     

Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Methylococcus capsulatus (Bath) uses a soluble methane monooxygenase (sMMO) to catalyse the oxidation of methane to methanol. sMMO is comprised of three components; A, B and C. Protein C (the reductase) transfers electrons from NADH to protein A (the hydroxylase) which contains the active site, and protein B regulates this electron flow. The five genes encoding the sMMO proteins and their subunits are clustered and have been cloned in Escherichia coli. A DNA fragment containing mmoB, the gene encoding protein B, was subcloned into pT7-5, a plasmid of the T7 RNA polymerase promoter expression system. Upon induction, E. coli expressed protein B which was fully functional after purification. The gene encoding protein C, mmoC, was amplified with unique restriction sites at each end using the polymerase chain reaction and then subcloned into pT7-7 (a plasmid similar to pT7-5 but containing its own ribosome-binding site and ATG start codon). Protein C expressed in E. coli was also found to be functional. This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in our analysis of the assembly and catalysis of sMMO.     

Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath)

Aerobic stopped-flow experiments have confirmed that component C is the methane monooxygenase component responsible for interaction with NADH. Reduction of component C by NADH is not the rate-limiting step for component C in the methane monooxygenase reaction. Removal and reconstitution of the redox centres of component C suggest a correlation between the presence of the FAD and Fe2S2 redox centres and NADH: acceptor reductase activity and methane monooxygenase activity respectively, consistent with the order of electron flow: NADH----FAD----Fe2S2----component A. This order suggests that component C functions as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for transfer to component A via the one-electron-carrying Fe2S2 centre. Electron transfer has been demonstrated between the reductase component, component C and the oxygenase component, component A, of the methane monooxygenase complex from Methylococcus capsulatus (Bath) by three separate methods. This intermolecular electron transfer step is not rate-determining for the methane monooxygenase reaction. Intermolecular electron transfer was independent of component B, the third component of the methane monooxygenase. Component B is required to switch the oxidase activity of component A to methane mono-oxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.     

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。     

Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b

The oxidation of methane to methanol in methanotrophic bacteria is catalysed by the enzyme methane monooxygenase (MM0). This multicomponent enzyme catalyses a range of oxidations including that of aliphatic and aromatic compounds and therefore has potential for commercial exploitation. This study details the molecular characterization of the soluble MMO (sMMO) genes from the Type II methanotroph Methylosinus trichosporium OB3b. The structural genes encoding the alpha, beta and gamma subunits of sMMO protein A and the structural gene encoding component B have been isolated and sequenced. These genes have been expressed and their products identified using an in vitro system. A comparative analysis of sMMO predicted sequences of M. trichosporium OB3b and the taxonomically related M. capsulatus (Bath) is also presented.     

Redox properties of the hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath). Effects of protein B, reductase, and substrate.

The reduction potentials of the hydroxylase component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been investigated through potentiometric titrations. The potentials were determined by EPR spectroscopic quantitation of the mixed valent hydroxylase as a function of added sodium dithionite in the presence of appropriate mediators. The reduction of the oxidized Fe(III).Fe(III) form to the mixed valent Fe(II).Fe(III) form occurs at 48 mV versus NHE while the potential for the formation of the fully reduced Fe(II).Fe(II) species from the mixed valent form was determined to be -135 mV. Addition of the substrate propylene to the hydroxylase did not have a major effect on the reduction potentials. Introduction of the protein B and the reductase components, however, completely inhibited reduction of the hydroxylase at potentials as far negative as -200 mV. Addition of propylene to all three methane monooxygenase components greatly facilitated hydroxylase reduction. Under these conditions, the fully reduced form of the protein was obtained at potentials of greater than 150 mV. This high redox potential indicates that the oxidized form of the protein is highly reactive, as required for methane oxidation. The present results reveal aspects of how both protein B and substrate can regulate electron transfer into and out of the hydroxylase component of methane monooxygenase.     

Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.     

Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.     

Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M

The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out.     

Detection of methanotrophic bacteria in environmental samples with the PCR.

We designed PCR primers by using the DNA sequences of the soluble methane monooxygenase gene clusters of Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and these primers were found to be specific for four of the five structural genes in the soluble methane monooxygenase gene clusters of several methanotrophs. We also designed primers for the gram-negative methylotroph-specific methanol dehydrogenase gene moxF. The specificity of these primers was confirmed by hybridizing and sequencing the PCR products obtained. The primers were then used to amplify methanotroph DNAs in samples obtained from various aquatic and terrestrial environments. Our sequencing data suggest that a large number of different methanotrophs are present in peat samples and also that there is a high level of variability in the mmoC gene, which codes for the reductase component of the soluble methane monooxygenase, while the mmoX gene, which codes for the alpha subunit of the hydroxylase component of this enzyme complex, appears to be highly conserved in methanotrophs.     

Solubilisation of methane monooxygenase from Methylococcus capsulatus (Bath)

The membrane-bound (particulate) form of methane monooxygenase from Methylococcus capsulatus (Bath) has been solubilised using the non-ionic detergent dodecyl-beta-D-maltoside. A wide variety of detergents were tested and found to solubilise membrane proteins but did not yield methane monooxygenase in a form that could be subsequently activated. After solubilisation with dodecyl-beta-D-maltoside, enzyme activity was recovered using either egg or soya-bean lipids. Attempts to further purify the solubilized methane monooxygenaser protein into its component polypeptides were unsuccessful and resulted in complete loss of enzyme activity. The major polypeptides present in the solubilised enzyme had molecular masses of 49 kDa, 23 kDa and 22 kDa which were similar to those seen in crude extracts [Prior, S. D. & Dalton H. (1985) J. Gen. Microbiol. 131, 155-163]. Studies on substrate and inhibitor specificities indicated that the membrane-associated and solubilised forms of methane monooxygenase were quite similar to each other but differed substantially from the well-characterised soluble methane monooxygenase found in cells grown in a low copper regime and synthesised independently of the particulate methane monooxygenase.     

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.     

油气田土壤DNA提取方法及油气指示菌基因定量结果的比较

采用4种提取方法对油田上方6个土壤样品微生物总基因组DNA进行提取,并比较了其纯度和浓度。利用定量PCR技术定量分析各土壤中甲烷单加氧酶基因（pmoA）和丁烷单加氧酶基因（brnoX）。与DNA试剂盒法相比,玻璃珠击打法、液氮研磨法、反复冻融法得到DNA纯度较低,需纯化才能进行后续的PCR扩增。液氮研磨法得到的DNA完整性、纯度和得率较其他提取方法均较好,尤其对于生物量较少的深层油田土壤DNA提取较为实用,成本较低。甲烷单加氧酶基因（pmoA）和丁烷单加氧酶基因（bmoX）的定量PCR结果表明,液氮研磨法提取DNA样品的定量结果在气区和油区均出现一定的高值。液氮研磨法较其他方法更适合于油田土壤DNA的提取。下一步研究可以把丁烷氧化茵作为油气指示茵研究中的重点检测对象。     

Evolution of the soluble diiron monooxygenases

Based on structural, biochemical, and genetic data, the soluble diiron monooxygenases can be divided into four groups: the soluble methane monooxygenases, the Amo alkene monooxygenase of Rhodococcus corallinus B-276, the phenol hydroxylases, and the four-component alkene/aromatic monooxygenases. The limited phylogenetic distribution of these enzymes among bacteria, together with available genetic evidence, indicates that they have been spread largely through horizontal gene transfer. Phylogenetic analyses reveal that the alpha- and beta-oxygenase subunits are paralogous proteins and were derived from an ancient gene duplication of a carboxylate-bridged diiron protein, with subsequent divergence yielding a catalytic alpha-oxygenase subunit and a structural beta-oxygenase subunit. The oxidoreductase and ferredoxin components of these enzymes are likely to have been acquired by horizontal transfer from ancestors common to unrelated diiron and Rieske center oxygenases and other enzymes. The cumulative results of phylogenetic reconstructions suggest that the alkene/aromatic monooxygenases diverged first from the last common ancestor for these enzymes, followed by the phenol hydroxylases, Amo alkene monooxygenase, and methane monooxygenases.     

Pacific Northwest marine sediments contain ammonia-oxidizing bacteria in the beta subdivision of the Proteobacteria

The diversity of ammonia-oxidizing bacteria in aquatic sediments was studied by retrieving ammonia monooxygenase and methane monooxygenase gene sequences. Methanotrophs dominated freshwater sediments, while beta-proteobacterial ammonia oxidizers dominated marine sediments. These results suggest that gamma-proteobacteria such as Nitrosococcus oceani are minor members of marine sediment ammonia-oxidizing communities.     

Isolation and characterization of methane utilizing bacteria from wetland paddy ecosystem

Methylotrophic bacteria which are known to utilize C1 compounds including methane. Research during past few decades increased the interest in finding out novel genera of methane degrading bacteria to efficiently utilize methane to decrease global warming effect. Moreover, evaluation of certain known plant growth promoting strains for their methane degrading potential may open up a new direction for multiple utility of such cultures. In this study, efficient methylotrophic cultures were isolated from wetland paddy fields of Gujarat. From the overall morphological, biochemical and molecular characterization studies, the isolates were identified and designated as Bacillus aerius AAU M 8; Rhizobium sp. AAU M 10; B. subtilis AAU M 14; Paenibacillus illinoisensis AAU M 17 and B. megaterium AAU M 29. Gene specific PCR analysis of the isolates, P. illinoisensis, B. aerius, Rhizobium sp. and B. subtilis showed presence of pmoA gene encoding α subunit particulate methane monooxygenase cluster. B. megaterium, P. illinoisensis, Rhizobium sp. and Methylobacterium extrorquens showed presence of mmoX gene encoding α subunit of the hydroxylase component of the soluble methane monooxygenase cluster. P. illinoisensis and Rhizobium sp. showed presence mxaF gene encoding α subunit region of methanol dehydrogenase gene cluster showing that both isolates are efficient utilizers of methane. To the best of our knowledge, this is the first time report showing presence of methane degradation enzymes and genes within the known PGPB group of organisms from wet land paddy agro-ecosystem, which is considered as one of the leading methane producer.     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶还原酶组分的纯化和性质

Ｍｅｔｙｌｏｍｏｎａｓｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析、ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌＨＰＬＣ分离纯化出ＭＭＯ还原酶组分．经ＨＰＬＣ分析,纯度大于９５％,纯化倍数为４．４,加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白．ＳＤＳ－ＰＡＧＥ电泳表明还原酶由一种亚基组成,分子量４２ｋＤ．ＩＣＰ－ＡＥＳ测定还原酶的Ｆｅ含量为１．８３ｍｏｌＦｅ每ｍｏｌ蛋白．ＵＶ－Ｖｉｓ光谱表明还原酶除２８０ｎｍ蛋白质特征峰外在４６０ｎｍ有最大吸收峰,且Ａ２８０ｎｍ／Ａ４６０ｎｍ为２．５０,与其它黄素一铁硫蛋白相似,推测还原酶可能含一个ＦＡＤ辅基和Ｆｅ２Ｓ２中心．在厌氧条件下,还原酶能够和ＮＡＤＨ作用,ＵＶ－Ｖｉｓ光谱分析表明还原酶４６０ｎｍ处特征吸收峰消失,说明在ＭＭＯ催化过程中还原酶接受ＮＡＤＨ的电子．ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析分离出调节蛋白Ｂ,部分纯化的调节蛋白Ｂ的分子量大约在２０ｋＤ,它能够提高ＭＭＯ比活性４０倍,ＭＭＯ还原酶和调节蛋白Ｂ单独存在时不具有ＭＭＯ