Chloromethane stimulates growth of Methylomicrobium album BG8 on methanol

Studies were performed to determine if the growth of Methylomicrobium album BG8 on methanol could be enhanced through the provision of chloromethane. M. album BG8 was found to be able to oxidize chloromethane via the particulate methane monooxygenase with an apparent K(s) of 11+/-3 microM and V(max) of 15+/-0.6 nmol (min mg protein)(-1). When up to 2.6 mM chloromethane was provided with 5 mM methanol, methanotrophic growth was significantly enhanced in comparison to the absence of chloromethane, indicating that methanotrophs can utilize chloromethane to support growth, although it could not serve as a sole growth substrate. [(14)C]chloromethane was found to be oxidized to [(14)C]CO(2) as well as incorporated into biomass. These results indicate that reactions previously thought to be cometabolic may actually provide some benefit to methanotrophs and that these cells can use multiple compounds to enhance growth.     

Degradation of chlorinated and brominated hydrocarbons by Methylomicrobium album BG8

The degradation kinetics of ten halogenated hydrocarbons by Methylomicrobium album BG8 expressing particulate methane monooxygenase (pMMO) and the inhibitory effects of these compounds on microbial growth and whole-cell pMMO activity were measured. When M. album BG8 was grown with methane, growth was completely inhibited by dichloromethane (DCM), bromoform (BF), chloroform (CF), vinyl chloride (VC), 1,1-dichloroethylene (1,1-DCE), and cis-dichloroethylene (cis-DCE). Trichloroethylene (TCE) partially inhibited growth on methane, while dibromomethane (DBM), trans-dichloroethylene (trans-DCE), and 1,1,1-trichloroethane (1,1,1-TCA) had no effect. If the cells were grown with methanol, DCM, BF, CF, and 1,1-DCE completely inhibited growth, while VC, trans-DCE, TCE, and 1,1,1-TCA partially inhibited growth. Both DBM and cis-DCE had no effect on growth with methanol. Whole-cell pMMO activity was also affected by these compounds, with all but 1,1,1-TCA, DCM, and DBM reducing activity by more than 25%. DCM, DBM, VC, trans-DCE, cis-DCE, 1,1-DCE, and TCE were degraded and followed Michaelis-Menten kinetics. CF, BF, and 1,1,1-TCA were not measurably degraded. These results suggested that the products of DCM, TCE, VC, and 1,1-DCE inactivated multiple enzymatic processes, while trans-DCE oxidation products were also toxic but to a lesser extent. cis-DCE toxicity, however, appeared to be localized to pMMO. Finally, DBM and 1,1,1-TCA were not inhibitory, and CF and BF were themselves toxic to M. album BG8. Based on these results, the compounds could be separated into four general categories, namely (1) biodegradable with minimal inactivation, (2) biodegradable with substantial inactivation, (3) not biodegradable with minimal inactivation, and (4) not biodegradable but substantial inactivation of cell activity. Received: 17 June 1999 / Accepted: 3 September 1999     

Isolation of Intracytoplasmic Membrane from the Methanotrophic Bacterium Methylomicrobium album BG8

Methane-oxidizing bacteria, including Methylomicrobium album BG8, form an intracytoplasmic membrane in addition to the cytoplasmic and outer membranes of the cell envelope. Techniques to isolate the intracytoplasmic membrane of M. album BG8 were developed. An intracytoplasmic membrane fraction was separated from a cell envelope fraction on the basis of sedimentation velocity in sucrose density gradients. Proteins associated with the particulate methane monooxygenase were found in both membrane fractions. Received: 27 July 1999 / Accepted: 30 August 1999     

Type 2 Cu2+ in pMMO from Methylomicrobium album BG8

EPR spectra were obtained for the type 2 Cu2+ site in particulate methane monooxygenase (pMMO) from Methylomicrobium album BG8 grown on K15NO3 and 63Cu(NO3)2. The concentration of the type 2 Cu2+ signal was approximately 200 microM per 25 mg/ml protein in packed cells and membrane fractions, a concentration that is consistent with its attribution to pMMO, and the EPR parameters were consistent with electron paramagnetic resonance (EPR) parameters previously assigned to pMMO. The superhyperfine structure due to nitrogen is better resolved because I = 1/2 for 15N whereas I = 1 for 14N and A(15N)/A(14N) = 1.4. Under these conditions, superhyperfine structure is resolved in the g region of the X-band spectrum. At low microwave frequency (S-band) the resolution of the nitrogen superhyperfine structure improves. Signals are attributed to type 2 Cu2+ in which cupric ion is bound to four (less likely three) nitrogen donor atoms.     

Soluble and particulate methane monooxygenase gene clusters in the marine methanotroph Methylomicrobium sp. strain NI

Soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO) gene clusters in the marine methanotroph Methylomicrobium sp. strain NI were completely sequenced and analysed. Degenerated primers were newly designed and used to amplify the gene fragments containing intergenic mmoX-Y and mmoD-C regions and a partial pmoC region. Phylogenetic analysis of amino acid sequences deduced from mmoX and pmoA, as well as of 16S rRNA gene sequences, indicated that this strain was most closely related to the halotolerant methanotroph Methylomicrobium buryatense. There were putative sigma(54)- and sigma(70)-dependent promoter sequences upstream of the sMMO and pMMO genes, respectively, and mmoG, which is known to be related to the expression and assembly of sMMO, existed downstream of the sMMO genes. These findings suggest that the major components and regulation of MMOs in this marine methanotroph are quite similar to those in freshwater methane oxidizers, despite the difference in their habitats.     

Comparison of EPR-visible Cu(2+) sites in pMMO from Methylococcus capsulatus (Bath) and Methylomicrobium album BG8

X-band (9.1 GHz) and S-band (3.4 GHz) electron paramagnetic resonance (EPR) spectra for particulate methane monooxygenase (pMMO) in whole cells from Methylococcus capsulatus (Bath) grown on (63)Cu and (15)N were obtained and compared with previously reported spectra for pMMO from Methylomicrobium album BG8. For both M. capsulatus (Bath) and M. album BG8, two nearly identical Cu(2+) EPR signals with resolved hyperfine coupling to four nitrogens are observed. The EPR parameters for pMMO from M. capsulatus (Bath) (g( parallel) = 2.244, A( parallel) = 185 G, and A(N) = 19 G for signal one; g( parallel) = 2.246, A( parallel) = 180 G, and A(N) = 19 G for signal two) and for pMMO from M. album BG8 (g( parallel) = 2.243, A( parallel) = 180 G, and A(N) = 18 G for signal one; g( parallel) = 2. 251, A( parallel) = 180 G, and A(N) = 18 G for signal two) are very similar and are characteristic of type 2 Cu(2+) in a square planar or square pyramidal geometry. In three-pulse electron spin echo envelope modulation (ESEEM) data for natural-abundance samples, nitrogen quadrupolar frequencies due to the distant nitrogens of coordinated histidine imidazoles were observed. The intensities of the quadrupolar combination bands indicate that there are three or four coordinated imidazoles, which implies that most, if not all, of the coordinated nitrogens detected in the continuous wave spectra are from histidine imidazoles.     

Architecture and active site of particulate methane monooxygenase

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O₂ binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies.     

Structural conservation of the B subunit in the ammonia monooxygenase/particulate methane monooxygenase superfamily

The ammonia monooxygenase (AMO)/particulate methane monooxygenase (pMMO) superfamily is a diverse group of membrane‐bound enzymes of which only pMMO has been characterized on the molecular level. The pMMO active site is believed to reside in the soluble N‐terminal region of the pmoB subunit. To understand the degree of structural conservation within this superfamily, the crystal structure of the corresponding domain of an archaeal amoB subunit from Nitrosocaldus yellowstonii has been determined to 1.8 Å resolution. The structure reveals a remarkable conservation of overall fold and copper binding site location as well as several notable differences that may have implications for function and stability. Proteins 2014; 82:2263–2267. © 2014 Wiley Periodicals, Inc.     

Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase

Rice field soil with a nonsaturated water content induced CH4 consumption activity when it was supplemented with 5% CH4. After a lag phase of 3 days, CH4 was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH4 mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the gamma subdivision of the class Proteobacteria [gamma-Proteobacteria]) and type II methylotrophs (members of the alpha-Proteobacteria) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH4 was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera Flavobacterium and Bacillus and was not influenced by CH4. The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera Methylobacter, Methylococcus, and Methylocystis. DGGE profiles of PCR products amplified with functional gene primer sets that targeted the mxaF and pmoA genes revealed that there were pronounced community shifts when CH4 oxidation began. High CH4 concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.     

Theoretical modeling of the hydroxylation of methane as mediated by the particulate methane monooxygenase

We present here the results of density functional theory (DFT) calculations directed toward elucidation of the CH bond activation mechanism that might be adopted by the particulate methane monooxygenase (pMMO) in the hydroxylation of methane and related small alkanes. In these calculations, we considered three of the most probable models for the transition metal active site mediating the "oxo-transfer": (i) the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1, recently proposed by Chan et al. [S.I. Chan, K.H.-C. Chen, S.S.-F. Yu, C.-L. Chen, S.S.-J. Kuo, Biochemistry 43 (2004) 4421-4430.]; (ii) the most frequently used model complex, bis(mu-oxo)Cu(III)(2) complex 2; and (iii) the mixed-valence bis(mu-oxo)Cu(II)Cu(III) complex 3. The results obtained indicate that the methane hydroxylation chemistry mediated by the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1 offers the most facile pathway for methane hydroxylation, and this model yields KIE values that are in good agreement with experiment. In this mechanism, the reaction proceeds along a "singlet" potential surface and a "singlet oxene" is directly inserted across a CH bond in a concerted manner. Kinetic isotope effects (k(H)/k(D) or KIE) associated with the concerted oxene insertion process mediated by complex 1 are calculated to be 5.2 at 300K when tunneling effects are included. Overall rate constants for the methane hydroxylation by the three models have been calculated as a function of temperature, and the rates are at least 5-6 orders of magnitude more facile when the chemistry is mediated by complex 1 compared to complex 2 or complex 3.     

颗粒状甲烷单加氧酶异源表达方法

由于甲烷氧化菌只能利用甲烷作为唯一的碳源和能源,存在生长缓慢、细胞密度低、培养困难等问题,限制了其工业应用。解决该问题的有效途径之一是在容易实现高密度培养的异源宿主菌中表达甲烷单加氧酶(Methane monooxygenase,MMO)。本实验室前期首次在一种红球菌中成功地表达了来自于甲烷氧化菌(Methylosinus trichosporium)OB3b的pMMO(颗粒状甲烷单加氧酶),但比酶活较原始菌低很多。本实验在该结果的基础上,通过选用不同的启动子和宿主细胞探索表达pMMO的可能性,结果得到了具有氧化甲烷活性的重组菌,但是产物检测到乙醇的生成,且该重组菌的pMMO活性不稳定,暗示pMMO的催化特性可能发生了变化。另外,很多重组菌检测到pMMO蛋白的表达,但没有催化活性,说明pMMO在宿主细胞中的正确组装是其功能表达的关键。     

Oxidation of methane by Methylomicrobium album and Methylocystis sp. in the presence of H2S and NH3

Oxidation of methane by methanotrophs, Methylomicrobium album and Methylocystis sp., was measured at several initial concentrations of H₂S and NH₃ in the headspace of stoppered flasks, at the same initial concentration of methane as sole carbon and energy source: 15 % (v/v). No effect was observed at 0.01 % (v/v) H₂S and 0.025 % (v/v) NH₃ in gas phase but over 0.05 and 0.025 % (v/v), respectively, they inhibited the oxidation of methane. The effect of H₂S was stronger in Methylocystis sp. and both microorganisms were similarly affected by NH₃. Depending on their concentrations in gas phase, H₂S and NH₃ can thus affect the rate of oxidation of methane and biomass growth of both methanotrophs.     

Polarized ATR-FTIR spectroscopy of the membrane-embedded domains of the particulate methane monooxygenase

The particulate methane monooxygenase (pMMO) of Methylococcus capsulatus (Bath) is an integral membrane protein that catalyzes the conversion of methane to methanol. To gain some insight into the structure-reactivity pattern of this protein, we have applied attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to investigate the secondary structure of the pMMO. The results showed that ca. 60% of the amino acid residues were structured as alpha-helices. About 80% of the peptide residues were estimated to be protected from the amide (1)H/(2)H exchange during a 21 h exposure to (2)H(2)O. In addition, a significant portion of the protein was shown to be sequestered within the bilayer membrane, protected from trypsin proteolysis. The ATR-FTIR difference spectrum between the intact and the proteolyzed pMMO-enriched membranes revealed absorption peaks only in the spectral regions characteristic for unordered and beta-structures. These observations were corroborated by amino acid sequence analysis of the pMMO subunits using the program TransMembrane topology with a Hidden Markov Model: 15 putative transmembrane alpha-helices were predicted. Finally, an attempt was also made to model the three-dimensional folding of the protein subunits from the sequence using the Protein Fold Recognition Server based on the 3D Position Specific Scoring Matrix Method. The C-terminal solvent-exposed sequence (N255-M414) of the pMMO 45 kDa subunit was shown to match the beta-sheet structure of the multidomain cupredoxins. We conclude on the basis of this ATR-FTIR study that pMMO is an alpha-helical bundle with ca. 15 transmembrane alpha-helices embedded in the bilayer membrane, together with a water-exposed domain comprised mostly of beta-sheet structures similar to the cupredoxins.     

Intracellular localization and characteristics of monophenol monooxygenase in the leaves of tea plants

The intracellular localization and some properties of monophenol monooxygenase (MPMO) from fresh tea leaves have been studied. It has been demonstrated that MPMO activity is located in cytosole and chloroplasts. These two forms have different properties. Molecular weights of cytosole and chloroplasts MPMO are 41 and 28 kD respectively. The chloroplasts and cytosole forms of MPMO reveal maximum activity at pH 5.3 and 7.1 respectively.     

Toward delineating the structure and function of the particulate methane monooxygenase from methanotrophic bacteria

The particulate methane monooxygenase (pMMO) is a complex membrane protein complex that has been difficult to isolate and purify for biochemical and biophysical characterization because of its instability in detergents used to solubilize the enzyme. In this perspective, we summarize the progress recently made toward obtaining a purified pMMO-detergent complex and characterizing the enzyme in pMMO-enriched membranes. The purified pMMO is a multi-copper protein, with ca. 15 copper ions sequestered into five trinuclear copper clusters: two for dioxygen chemistry and alkane hydroxylation (catalytic or C-clusters) and three to provide a buffer of reducing equivalents to re-reduce the C-clusters following turnover (electron transfer or E-clusters). The enzyme is functional when all the copper ions are reduced. When the protein is purified under ambient aerobic conditions in the absence of a hydrocarbon substrate, only the C-clusters are oxidized; there is an apparent kinetic barrier for electron transfer from the E-cluster copper ions to the C-clusters under these conditions. Evidence is provided in support of both C-clusters participating in the dioxygen chemistry, but only one C-cluster supporting alkane hydroxylation. Acetylene modification of the latter C-cluster in the hydrophobic pocket of the active site lowers or removes the kinetic barrier for electron transfer from the E-clusters to the C-clusters so that all the copper ions could be fully oxidized by dioxygen. A model for the hydroxylation chemistry when a hydrocarbon substrate is bound to the active site of the hydroxylation C-cluster is presented. Unlike soluble methane monooxygenase (sMMO), pMMO exhibits limited substrate specificity, but the hydroxylation chemistry is highly regioselective and stereoselective. In addition, the hydroxylation occurs with total retention of configuration of the carbon center that is oxidized. These results are consistent with a concerted mechanism involving direct side-on insertion of an active singlet "oxene" from the activated copper cluster across the "C-H" bond in the active site. Finally, in our hands, both the purified pMMO-detergent complex and pMMO-enriched membranes exhibit high NADH-sensitive as well as duroquinol-sensitive specific activity. A possible role for the two reductants in the turnover of the enzyme is proposed.     

Biological methane oxidation: regulation, biochemistry, and active site structure of particulate methane monooxygenase

Particulate methane monooxygenase (pMMO) is a three-subunit integral membrane enzyme that catalyzes the oxidation of methane to methanol. Although pMMO is the predominant methane oxidation catalyst in nature, it has proved difficult to isolate, and most questions regarding its molecular structure, active site composition, chemical mechanism, and genetic regulation remain unanswered. Copper ions are believed to play a key role in both pMMO regulation and catalysis, and there is some evidence that the enzyme contains iron as well. A number of research groups have solubilized and purified or partially purified pMMO. These preparations have been characterized by biochemical and biophysical methods. In addition, aspects of methane monooxygenase gene regulation and copper accumulation in methanotrophs have been studied. This review summarizes for the first time the often controversial pMMO literature, focusing on recent progress and highlighting unresolved issues.     

Intracellular localization of aldehyde dehydrogenase in rat liver

1. Distribution of aldehyde dehydrogenase activity in rat liver was studied by measuring the rate of disappearance of acetaldehyde in the presence of each of the subcellular fractions. These were obtained by rough separation of particulate fractions from the soluble portion of the cell, by differential centrifugation, and by isopycnic gradient centrifugation. 2. The maximal rate of acetaldehyde oxidation was 3.7 mumol/min per g, with an apparent K(m) value below 10(-5)m. The highest rate of activity was observed in phosphate buffers of high P(i) concentration (above 60mm). 3. The activity measured was completely dependent on NAD(+). 4. The microsomal fraction and the nuclei were inactive in the assay. Of the total activity 80% was found in the mitochondrial fraction and the remaining 20% in the cytoplasm. 5. The distribution pattern is important from the point of view of acetaldehyde oxidation during ethanol metabolism. The apparent discrepancy of the results obtained by different workers and the localization of acetaldehyde oxidation in vivo is discussed.