Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Stainthorpe, A. C.; Murrell, J. Colin; Salmond, George P. C.; Dalton, Howard; Lees, Veronica

doi:10.1007/bf00456094

Cited by 65 publications

(50 citation statements)

References 31 publications

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“…2. The mmo genes, which code for all five components of the MMO system, are contained on a 5.5-kb section of pCH4 (35). Two variations (Fd1 and Fd2) of the Fd portion of the mmoC gene, which encodes MMOR, were amplified from pCH4 with Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) by using the primers LKFB10 (Fd1; 5Ј-CTGCGGATCCACACTATCACGGCGGTG) or LKFB20 (Fd2; 5Ј-AGTTGGATCCGGCCATGTTAGCGGTTATTCG-CGAGTTCACACTATCACGGC) and FdRH10 (5Ј-CCTCTCCAAAGCT-TATGCGTCAATGGGTATAG).…”

Section: Construction Of Ptrc-fd1 and Ptrc-fd2mentioning

confidence: 99%

Domain Engineering of the Reductase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath)

Blazyk

Lippard

2004

Journal of Biological Chemistry

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The first step in the catabolic pathway of methanotrophs is the conversion of methane to methanol by soluble or membrane-bound methane monooxygenases (MMOs) 1 (1, 2). Although the soluble MMO (sMMO) system is expressed by only a few organisms under low copper conditions (3), it has been characterized far more extensively than the particulate form because of difficulties purifying and stabilizing the particulate MMO enzymes (4 -6). The sMMO proteins from two methanotrophic bacteria, Methylococcus capsulatus (Bath) (7,8) and Methylosinus trichosporium OB3b (9, 10), have been studied in great detail (11)(12)(13)(14)(15)(16)(17)(18)(19). Both systems require three components: a dimeric (␣␤␥) 2 hydroxylase (MMOH, 251 kDa) with dinuclear, carboxylate-bridged iron centers, where dioxygen activation and methane hydroxylation occur; a regulatory protein (MMOB, 15.9 kDa); and a reductase (MMOR, 38.5 kDa) that transfers electrons from NADH to the MMOH diiron sites, thereby priming the hydroxylase for reaction with dioxygen. The N-terminal domain of MMOR, which houses a [2Fe-2S] cluster, shares sequence (20), spectroscopic (16,(21)(22)(23)(24), and structural (25) properties with ferredoxins of plants, cyanobacteria, and archaebacteria. A flavin adenine dinucleotide (FAD) cofactor located in the C-terminal portion of MMOR accepts two electrons from NADH. These electrons are passed sequentially from the reduced FAD moiety through the MMOR [2Fe-2S] center into the hydroxylase active sites by means of a series of intra-and intermolecular electron transfer reactions (16,24,26,27).MMOR is a member of a class of modular flavoprotein electron transferases, also known as the ferredoxin:NADP ϩ oxidoreductase (FNR) family. These proteins contain a flavin domain that transfers electrons between a nicotinamide dinucleotide and a one-electron carrier domain, which may be linked or dissociable (28 -30). Because the electron carrier domain can be attached to either the N-or C-terminal end of the core flavin/NAD(P) unit, this domain appears to be an independent modular element. In support of this designation, the crystal structure of Burkholderia cepacia phthalate dioxygenase reductase (PDR), an FNR family member with C-terminal electron carrier connectivity, shows distinct flavin mononucleotide (FMN), NADH binding, and [2Fe-2S] domains ( Fig. 1) (30). To examine the modular nature of MMOR and facilitate characterization of this complex protein, the ferredoxin (MMOR-Fd) and FAD/NADH (MMOR-FAD) domains were expressed as separate polypeptides (31). NMR studies reveal that * This work was supported by National Institutes of Health Research Grant GM32134 (to S. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ Howard Hughes Medical Institute predoctoral fellow. § To whom correspondence should be addressed.

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Section: Construction Of Ptrc-fd1 and Ptrc-fd2mentioning

confidence: 99%

Domain Engineering of the Reductase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath)

Blazyk

Lippard

2004

Journal of Biological Chemistry

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“…The sMMO enzyme, however, is only found in some methanotrophs, and the most extensively characterized sMMO enzymes are those from Methylococcus capsulatus (Bath) (Colby & Dalton, 1976;Green & Dalton, 1985;Woodland & Dalton, 1984) and Ms. trichosporium OB3b (Fox et al, 1989). The purified enzyme is composed of a threecomponent hydroxylase (abc) 2 encoded by mmoXYZ, a reductase encoded by mmoC, and a regulatory protein, protein B, encoded by mmoB (Cardy et al, 1991a, b;Pilkington & Dalton, 1991;Stainthorpe et al, 1989). The a subunit of the hydroxylase contains a binuclear iron centre, which is essential for catalysis (Elango et al, 1997;Rosenzweig et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis

et al. 2006

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The soluble methane monooxygenase (sMMO) is a key enzyme for methane oxidation, and is found in only some methanotrophs, including Methylosinus sporium 5. sMMO expression is regulated at the level of transcription from a σ 54 promoter by a copper-switch, and is only expressed when the copper-to-biomass ratio during growth is low. Extensive phylogenetic and genetic analyses of sMMOs and other soluble di-iron monooxygenases reveal that these enzymes have only been acquired relatively recently through horizontal gene transfer. In this study, further evidence of horizontal gene transfer was obtained, through cloning and sequencing of the genes encoding the sMMO enzyme complex plus the regulatory genes mmoG and mmoR, and identification of a duplicate copy of the mmoX gene in Ms. sporium. mmoX encodes the α subunit of the hydroxylase of the sMMO enzyme, which constitutes the active site (Prior & Dalton, 1985). The mmoX genes were characterized at the molecular and biochemical levels. Although both copies were transcribed, only mmoX copy 1 was essential for sMMO activity. Construction of an sMMO− mutant by marker-exchange mutagenesis gave some possible insights into the role of the water-soluble pigment in siderophore-mediated iron acquisition. Finally, the amenability of Ms. sporium to genetic manipulation was demonstrated by complementing the sMMO− mutant by heterologous expression of sMMO genes from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and it was shown that Ms. sporium could be used as an alternative model organism for molecular analysis of MMO regulation.

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“…The 5?4 kb sequence downstream from mmoC was already cloned on pCH4 (Stainthorpe et al, 1989(Stainthorpe et al, , 1990. pCH4 was digested with Bsp120I and the resulting 1?1 and 4?2 kb fragments were isolated.…”

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Genes involved in the copper-dependent regulation of soluble methane monooxygenase of Methylococcus capsulatus (Bath): cloning, sequencing and mutational analysis

et al. 2003

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The key enzyme in methane metabolism is methane monooxygenase (MMO), which catalyses the oxidation of methane to methanol. Some methanotrophs, including Methylococcus capsulatus (Bath), possess two distinct MMOs. The level of copper in the environment regulates the biosynthesis of the MMO enzymes in these methanotrophs. Under low-copper conditions, soluble MMO (sMMO) is expressed and regulation takes place at the level of transcription. The structural genes of sMMO were previously identified as mmoXYBZ, mmoD and mmoC. Putative transcriptional start sites, containing a s 70 -and a s N -dependent motif, were identified in the 59 region of mmoX. The promoter region of mmoX was mapped using truncated 59 end regions fused to a promoterless green fluorescent protein gene. A 9?5 kb region, adjacent to the sMMO structural gene cluster, was analysed. Downstream (39) from the last gene of the operon, mmoC, four ORFs were found, mmoG, mmoQ, mmoS and mmoR. mmoG shows significant identity to the large subunit of the bacterial chaperonin gene, groEL. In the opposite orientation, two genes, mmoQ and mmoS, showed significant identity to two-component sensor-regulator system genes. Next to mmoS, a gene encoding a putative s N -dependent transcriptional activator, mmoR was identified. The mmoG and mmoR genes were mutated by marker-exchange mutagenesis and the effects of these mutations on the expression of sMMO was investigated. sMMO transcription was impaired in both mutants. These results indicate that mmoG and mmoR are essential for the expression of sMMO in Mc. capsulatus (Bath).

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Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Cited by 65 publications

References 31 publications

Domain Engineering of the Reductase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath)

Domain Engineering of the Reductase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath)

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis

Genes involved in the copper-dependent regulation of soluble methane monooxygenase of Methylococcus capsulatus (Bath): cloning, sequencing and mutational analysis

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