[31] Methane monooxygenase from Methylosinus trichosporium OB3b

Fox, Brian G.; Froland, Wayne A.; Jollie, David; Lipscomb, John D.

doi:10.1016/0076-6879(90)88033-7

Cited by 137 publications

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“…The growth of M. trichosporium OB3b and purification of MMOH and MMOB were as reported previously (Fox et al, 1989(Fox et al, , 1990a. The specific activity at 23 "C of MMOB was 7,000 nmol/min/mg.…”

Section: Crystal Formmentioning

confidence: 79%

“…MMO exists in both a soluble (sMMO) and a particulate (pMMO) form (Dalton, 1980;Anthony, 1982). sMMO has been purified and characterized from the Type X methanotroph, Methylococcus capsulatus Bath (MMO Bath) (Dalton, 1980, 199 Dalton, 1984;Pilkington & Dalton, 1990), and several Type I1 methanotrophs including, Methylosinus trichosporium 0B3b (MMO OB3b) (Fox et al, 1989(Fox et al, , 1990a, and Methylobacterium sp. CRL-26 (Patel & Savas, 1987).…”

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Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

et al. 1997

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Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the 02-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type 11, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type I1 methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type I1 Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A and 2.7 8, resolution, respectively, both determined at 18 "C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H20), as well as both carboxylate oxygens of Glu a144. This bis-yhydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 "C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the p chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including Da209E, a ligand of one of the irons.

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Section: Crystal Formmentioning

confidence: 79%

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confidence: 99%

Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

et al. 1997

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“…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.…”

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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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“…The enzyme consists of three components : the hydroxylase, the reductase and a regulatory protein, component B. The p-OH bridged diiron site [3, 41 resides in the hydroxylase and it is the fully reduced, Fe(II)Fe(II), form which is catalytically active [5]. …”

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Further Evidence for Multiple Pathways in Soluble Methane‐Monooxygenase‐Catalysed Oxidations from the Measurement of Deuterium Kinetic Isotope Effects

Wilkins

Dalton

Samuel

et al. 1994

European Journal of Biochemistry

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The data from the deuterium isotope experiments in this study show that the primary kinetic isotope effect for methane oxidation catalysed by soluble methane monooxygenase from Methylococcus capsulatus (Bath) is very small, <2. In contrast, the primary kinetic isotope effect for -CH, group oxidation in toluene is large, > 7. A mechanistic pathway in which a substrate radical is formed from hydrogen atom abstraction by a ferry1 species is believed to operate for CH,, the toluene -CH, group and similar alkanes. Direct oxygen atom addition, rather than H atom abstraction, is indicated for aromatic ring oxidations in benzene and toluene and for styrene oxide formation from styrene. Thus, more than one mechanistic pathway appears to operate in soluble methanemonooxygenase-catalysed reactions and, in some cases, the pathway chosen may be dictated by the substrate.In the soluble methane-monooxygenase-catalysed oxidation of toluene the rates of: (a) substrate dissociation from the enzyme-substrate complex, (b) product formation and (c) product release (benzyl alcohol and p-cresol) from the enzyme-product complex are comparable in magnitude. Therefore all three of these steps are partially rate-determining in the soluble methane monooxygenase catalytic cycle for toluene oxidation.The active site of the enzyme soluble methane monooxygenase [l] is just one example of the bridged diiron motif now known to occur in a number of non-heme iron proteins of vastly different functions [2]. In the case of soluble methane monooxygenase the function of the diiron site is to activate 0, so that one of the oxygen atoms is inserted into a methane C-H bond to form methanol. The other oxygen atom ends up in the second product, water. The enzyme consists of three components : the hydroxylase, the reductase and a regulatory protein, component B. The p-OH bridged diiron site [3, 41 resides in the hydroxylase and it is the fully reduced, Fe(II)Fe(II), form which is catalytically active [5]. 25).National Institutes of Health.high-valent iron-oxo intermediate, forming a substrate radical which then rapidly reacts with iron-bound OH to produce the product [9-111. This mechanism is, in many respects, similar to that proposed for cytochrome P-450 (121. It has been shown that P-450-catalysed hydroxylation of toluene produces benzyl alcohol plus o-, m-and p-cresols with benzyl alcohol as the major product [13 1. With fully deuterated toluene the product ratio was reversed and the cresols dominated. We had earlier observed that catalysis of toluene oxidation by soluble methane monooxygenase produced benzyl alcohol and only p-cresol (not ortho and metu) [8,9, 141. Recently Rataj et al. [15] reported that benzyl alcohol alone and no p-cresol was produced in this oxidation of toluene using soluble methane monooxygenase from Methylosinus trichosporium and crude extract from Methylococcus cupsulutus (Bath). They were also unable to detect any styrene oxide when styrene was used as substrate, a product we had observed previously with the M. cupsulutus ...

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[31] Methane monooxygenase from Methylosinus trichosporium OB3b

Cited by 137 publications

References 10 publications

Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

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

Further Evidence for Multiple Pathways in Soluble Methane‐Monooxygenase‐Catalysed Oxidations from the Measurement of Deuterium Kinetic Isotope Effects

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