Production of high-quality particulate methane monooxygenase in high yields from Methylococcus capsulatus (bath) with a hollow-fiber membrane bioreactor

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 micro M. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is approximately 13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl beta-D-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an alphabetagamma protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.     

Batch cultivation of Methylosinus trichosporium OB3b: II. Production of particulate methane monooxygenase

The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 muM and remained constant upon further increases of the copper concentration to 120 muM. The cell growth rate (mu), on the other hand, decreased over the entire range (10-120 muM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 muM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.     

The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.     

颗粒性甲烷单加氧酶的研究

从甲基弯菌(Methylosinus trichosporium)IMV3011(简称M3011)的膜中分离纯化出颗粒性甲烷单加氧酶(Particulate Methane monooxygenase,简称pMMO)和NADH脱氢酶。Cu2+、不同外源电子给体、EDTA等对pMMO活性的都有影响;测定了pMMO分子量,以及其活性中心金属离子含量。对于M3011,培养基中Cu2+浓度对pMMO的生成和活性没有影响;但在分离纯化过程中对pMMO稳定性有明显影响。电镜结果显示,Cu2+浓度对pMMO的数量有较明显的影响。对于纯化的pMMO,对苯二酚仍是有效的电子供体,而NADH却是无效的电子供体。纯化过程中采用对苯二酚作为pMMO活性分析时的电子供体,排除了共纯化NADH脱氢酶的必要,有利于对pMMO活性中心进行深入研究。     

Improvement of the purification method for retaining the activity of the particulate methane monooxygenase from Methylosinus trichosporium OB3b

The purification method of particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was improved, and purified pMMO retained its activity with duroquinol as a reductant. n-Dodecyl-,d-maltoside was used for the solubilization of pMMO and Brij 58 was used for the purification for anion exchange chromatography. Compared to the original pMMO activity in the membrane fraction, 88% of the activity was now retained in the purified material. The purified pMMO monomer (94 kDa) contained only two copper atoms and did not contain iron. Both copper ions showed only a typical type II copper EPR signal with a superhyperfine structure at the g region, indicating that the type II copper ions play an important role as the active site of methane hydroxylation in pMMO.     

Properties of the membranes containing the particulate methane monooxygenase from Methylosinus trichosporium OB3b

The particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was partiallypurified and characterized by measuring the effects of reducing agents and additives, and the stability ofpMMO was studied. Duroquinol was a suitable reducing agent, and pMMO was stabilized by bovine serumalbumin (BSA). Among the additivies, the copper (II) ion stimulated pMMO at low concentration andinhibited at high concentration. The optimum conditions for pMMO activity were as follows: 45 ° C, pH 6.5and 55 mM 3-morpholinopropanesulfonic acid (MOPS) buffer, and the rate of propene epoxide formationwas 13.6 nmol min mg protein. ESR spectra indicate that the copper cluster in the membrane fraction isreduced by duroquinol and oxidized by dioxygen. The result suggests that the copper cluster is containedin the active site of pMMO.     

Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO(2) gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h(-1) and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 muM Cu, 80 muM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h(-1). Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 muM, falling sharply at 20 muM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. (c) 1992 John Wiley & Sons, Inc.     

The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.     

Biological Methane Oxidation: Regulation,Biochemistry, and Active Site Structure of Particulate Methane Monooxygenase

Particulate methane monooxygenase (pMMO) is a threesubunit 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.     

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.     

Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath).

An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide.     

Three-dimensional structure determination of a protein supercomplex that oxidizes methane to formaldehyde in 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). The active protein complex termed pMMO-C was purified recently from Methylococcus capsulatus (Bath). The complex consists of pMMO hydroxylase and an additional component pMMO-R, which was proposed to be the reductase for the pMMO complex. Further study of this complex has led here to the proposal that the pMMO-R is in fact methanol dehydrogenase, the subsequent enzyme in the methane oxidation pathway by methanotrophs. We describe here the biochemical and biophysical characterization of a stable purified complex of pMMO hydroxylase (pMMO-H) with methanol dehydrogenase (MDH) and report the first three-dimensional (3D) structure, determined by cryoelectron microscopy and single particle analysis to approximately 16 A resolution. The 3D structure reported here provides the first insights into the supramolecular organization of pMMO with MDH. These studies of pMMO-MDH complexes have provided further understanding of the structural basis for the particular functions of the enzymes in this system which might also be of relevance to the complete process of methane oxidation by methanotrophs under high copper concentration in the environment.     

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.     

Hydrogen peroxide as an effecter on the inactivation of particulate methane monooxygenase under aerobic conditions

Particulate methane monooxygenase (pMMO), a copper-containing membrane protein, catalyzes methane hydroxylation under aerobic conditions. We found that the activity of pMMO was increased by catalase, implying that hydrogen peroxide (H₂O₂) is generated by pMMO with duroquinol, an electron donor for pMMO, and that the generated H₂O₂ inhibits pMMO activity. In addition, reversible inhibition of pMMO with H₂O₂ was observed upon treatment of pMMO with H₂O₂ followed by the addition of catalase, and H₂O₂ formation by pMMO with duroquinol was detected using a fluorescence probe. The redox behavior of type 2 copper in pMMO measured by the electron paramagnetic resonance revealed that H₂O₂ re-oxidizes the type 2 copper in pMMO reduced with duroquinol.     

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.     

Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu(I) and Cu(II), of which the latter exhibits two distinct type 2 Cu(II) electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 ?. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 ? resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.     

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.     

Concentration of Cu, EPR-detectable Cu, and formation of cupric-ferrocyanide in membranes with pMMO

EPR spectra were obtained for the type 2 Cu(2+) site in particulate methane monooxygenase, pMMO, from membrane fractions of Methylomicrobium album BG8. In addition to the EPR signal with g parallel = 2.24 and A parallel = 185 G found in both cells and membrane fractions, a second EPR signal with g parallel = 2.29 and A parallel = 146 G was found in membrane fractions and attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu(2+) with total copper determined by atomic absorption suggests that there are two or three EPR-silent coppers for every EPR-detectable copper and that there are approximately four coppers per enzyme composed of the 47, 27, and 25 kDa subunits. Treatment of membrane fractions loaded with pMMO with Fe(CN)6(3-) results in a new EPR signal that is attributed to CuFe(CN)6(2-), not to an intrinsic trimeric copper cluster as previously reported in studies with a related bacterium.     

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.     

Functional expression of the particulate methane mono-oxygenase gene in recombinant Rhodococcus erythropolis

In order to construct an expression system for the particulate methane mono-oxygenase (pMMO) gene (pmo), the structural gene cluster pmoCAB amplified from Methylosinus trichosporium OB3b was inserted into a shuttle vector pBS305 under the control of a dsz promoter and transformed into Rhodococcus erythropolis LSSE8-1. A stable transformant was successfully obtained using ethane as the sole carbon source. Fluorescence in situ hybridization results showed that the dsz promoter allowed the pmo genes to be transcribed in the recombinant strain. The effects of Cu2+ and Zn2+ concentrations on cell growth and pMMO activity in ethane-containing medium were examined. It was discovered that 7.5 microM Cu2+ and 1.8 microM Zn2+ were suitable to achieve high cell concentration and pMMO activity, but the amount of methanol accumulated during methane oxidation by the recombinant strain was still low.