Particulate methane monooxygenase from Methylosinus trichosporium is a copper-containing enzyme

Xin, Jiaying; Cui, Jun-Ru; Hu, Xiaoxue; Li, Shuben; Xia, Chungu; Zhu, Li‐Min; Wang, Yiqun

doi:10.1016/s0006-291x(02)00647-2

Cited by 23 publications

(18 citation statements)

References 8 publications

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“…In addition to being a transcriptional switch, copper ions serve as a metabolic activator to produce large quantities of pMMO, as well as cell membranes to support the pMMO. In addition, since pMMO is a multicopper protein that requires many copper ions for biological function (4,15,16,17,23,24), there must be a threshold copper concentration in the growth medium during cell culturing before the expressed pMMO can incorporate the full complement of copper ions to assemble the fully functional enzyme. To illustrate this point, the copper uptake by the cells was examined by measuring the difference between the copper concentration of an applied fresh medium and the copper concentration remaining in the used medium after growth of the cell suspension to the mid-log phase.…”

Section: Resultsmentioning

confidence: 99%

“…In almost all of these studies (2,4,9,15,22,23,24,25), NADH activity was observed only in membranes, and the enzyme following detergent solubilization and purification by column chromatography exhibited activity only with duroquinol, usually with a significant loss of copper ions during the purification process. We believe that the fully functional protein was not obtained in these studies.…”

Section: Discussionmentioning

confidence: 99%

“…Three polypeptides with apparent molecular masses of 45, 27, and 23 kDa have been observed in the membrane fractions when M. capsulatus (Bath) switches from expressing the sMMO to expressing the pMMO. Addition of increasing amounts of copper ions to the growth medium leads to synthesis of additional intracytoplasmic membranes, increased expression of the pMMO, increased growth yields, and a concomitant loss of sMMO activity (15,19,24).…”

mentioning

confidence: 99%

“…When M. capsulatus (Bath) cells are cultured in a growth medium containing copper at an initial concentration higher than 40 M, extra copper ions are adsorbed by the membranes. This copper adsorption results in thickening of the cytoplasmic membrane (24) and eventually inactivates the cells. Thus, the optimal initial copper concentration for culture of this methanotroph should be around 30 to 35 M.…”

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confidence: 99%

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Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor

et al. 2003

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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 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 ϳ13 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 ␤-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 ␣␤␥ 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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor

et al. 2003

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“…In early work, Xin et al systematically explored the catalytic mechanism of whole cells and the characterization of pMMO, [20][21][22][23] but sMMO has not yet been purified and characterized. In order to confirm that sMMO is present in strain IMV 3011 and the different biochemical properties among sMMOs from different sources, this report focuses on identify and characterizing methane monooxygenase hydroxyy To whom correspondence should be addressed.…”

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confidence: 99%

Purification and Biochemical Characterization of Soluble Methane Monooxygenase Hydroxylase fromMethylosinus trichosporiumIMV 3011

Shi

Jia-yin

et al. 2007

Bioscience, Biotechnology, and Biochemistry

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Particulate Methane Monooxygenase

Rosenzweig

2004

Handbook of Metalloproteins

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Particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol in methanotrophic bacteria. The 300‐kDa pMMO enzyme is a trimeric integral membrane protein, comprising three copies each of three subunits: pmoB, pmoA, and pmoC. Purified pMMO contains both copper and iron, although some of the iron may be due to heme contamination. The copper stoichiometry ranges from 2 to 15 copper ions per 100‐kDa pMMO protomer, depending on the enzyme source. EPR spectra of whole cells, membrane‐bound, and purified pMMO consistently indicate the presence of type 2 Cu(II). According to XAS data, both Cu(I) and Cu(II) are present. EXAFS data reveal the presence of oxygen/nitrogen ligands and a short CuCu interaction at 2.5 Å that increases to 2.6 Å upon chemical reduction. Crystal structures of pMMO from two organisms have been reported. A soluble region consisting of six cupredoxin‐like β‐barrel structures is supported by 15 transmembrane helices. Three different metal centers have been detected crystallographically: a highly conserved dinuclear copper center, a nonconserved mononuclear copper center, and a site that can be occupied by zinc or copper. Although the identity of the pMMO active site remains unknown, some information regarding the mechanism has been obtained from substrate studies and computational work.

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Particulate methane monooxygenase from Methylosinus trichosporium is a copper-containing enzyme

Cited by 23 publications

References 8 publications

Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor

Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor

Purification and Biochemical Characterization of Soluble Methane Monooxygenase Hydroxylase fromMethylosinus trichosporiumIMV 3011

Particulate Methane Monooxygenase

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