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PubMed2004

NMR structure of the flavin domain from soluble methane monooxygenase reductase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to methanol, the first step in carbon assimilation by methanotrophs. This multicomponent system transfers electrons from NADH through a reductase component to the non-heme diiron center in the hydroxylase where O(2) is activated. The reductase component comprises three distinct domains, a [2Fe-2S] ferredoxin domain along with FAD- and NADH-binding domains. We report the solution structure of the reduced 27.6 kDa FAD- and NADH-binding domains (MMOR-FAD) of the reductase from Methylococcus capsulatus (Bath). The FAD-binding domain consists of a six-stranded antiparallel beta-barrel and one alpha-helix, with the first 10 N-terminal residues unstructured. In the interface between the two domains, the FAD cofactor is tightly bound in an unprecedented extended conformation. The NADH-binding domain consists of a five-stranded parallel beta-sheet with four alpha-helices packing closely around this sheet. MMOR-FAD is structurally homologous to other FAD-containing oxidoreductases, and we expect similar structures for the FAD/NADH-binding domains of reductases that occur in other multicomponent monooxygenases.

Amino Acid Sequence↗
PubMed2001

Why OrfY? Characterization of MMOD, a long overlooked component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) has been studied intensively to understand the mechanism by which it catalyzes the remarkable oxidation of methane to methanol. The cluster of genes that encode for the three characterized protein components of sMMO (MMOH, MMOB, and MMOR) contains an additional open reading frame (orfY) of unknown function. In the present study, MMOD, the protein encoded by orfY, was overexpressed as a fusion protein in Escherichia coli. Pure MMOD was obtained in high yields after proteolytic cleavage and a two-step purification procedure. Western blot analysis of Methylococcus capsulatus (Bath) soluble cell extracts showed that MMOD is expressed in the native organism although at significantly lower levels than the other sMMO proteins. The cofactorless MMOD protein is a potent inhibitor of sMMO activity and binds to the hydroxylase protein (MMOH) with an affinity similar to that of MMOB and MMOR. The addition of up to 2 MMOD per MMOH results in changes in the optical spectrum of the hydroxylase that suggest the formation of a (micro-oxo)diiron(III) center in a fraction of the MMOH-MMOD complexes. Possible functions for MMOD are discussed, including a role in the assembly of the MMOH diiron center similar to that suggested for DmpK, a protein that shares some properties with MMOD.

Amino Acid Sequence↗
PubMed1974

Purification and properties of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase from Methylococcus capsulatus.

3-Hexulose phosphate synthase and phospho-3-hexuloisomerase were purified 40- and 150-fold respectively from methane-grown Methylococcus capsulatus. The molecular weights of the enzymes were approximately 310000 and 67000 respectively, as determined by gel filtration. Dissociation of 3-hexulose phosphate synthase into subunits of molecular weight approx. 49000 under conditions of low pH or low ionic strength was observed. Within the range of compounds tested, 3-hexulose phosphate synthase is specific for formaldehyde and d-ribulose 5-phosphate (forward reaction) and d-arabino-3-hexulose 6-phosphate (reverse reaction), and phospho-3-hexuloisomerase is specific for d-arabino-3-hexulose 6-phosphate (forward reaction) and d-fructose 6-phosphate (reverse reaction). A bivalent cation is essential for activity and stability of 3-hexulose phosphate synthase; phospho-3-hexuloisomerase is inhibited by many bivalent cations. The pH optima of the two enzymes are 7.0 and 8.3 respectively and the equilibrium constants are 4.0x10(-5)m and 1.9x10(2)m respectively. The apparent Michaelis constants for 3-hexulose phosphate synthase are: d-ribulose 5-phosphate, 8.3x10(-5)m; formaldehyde, 4.9x10(-4)m; d-arabino-3-hexulose 6-phosphate, 7.5x10(-5)m. The apparent Michaelis constants for phospho-3-hexuloisomerase are: d-arabino-3-hexulose 6-phosphate, 1.0x10(-4)m; d-fructose 6-phosphate, 1.1x10(-3)m.

Aldehyde-Lyases↗
PubMed1999

Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath.

Genes for the subunits of particulate methane monooxygenase, PmoABC, have been sequenced from the gamma-proteobacterial methanotroph Methylococcus capsulatus Bath. M. capsulatus Bath contains two complete copies of pmoCAB, as well as a third copy of pmoC. The two pmoCAB regions were almost identical at the nucleotide sequence level, differing in only 13 positions in 3183 bp. At the amino acid level, each translated gene product contained only one differing residue in each copy. However, the pmoC3 sequence was more divergent from the two other pmoC copies at both the far N-terminus and far C-terminus. Chromosomal insertion mutations were generated in all seven genes. Null mutants could not be obtained for pmoC3, suggesting that it may play an essential role in growth on methane. Null mutants were obtained for pmoC1, pmoC2, pmoA1, pmoA2, pmoB1 and pmoB2. All of these mutants grew on methane, demonstrating that both gene copies were functional. Copy 1 mutants showed about two-thirds of the wild-type whole-cell methane oxidation rate, while copy 2 mutants showed only about one-third of the wild-type rate, indicating that both gene copies were necessary for wild-type particulate methane monooxygenase activity. It was not possible to obtain double null mutants that were defective in both pmo copies, which may indicate that some expression of pMMO is important for growth.

Amino Acid Sequence↗
PubMed1982

[Role of CO-binding cytochrome c in enzymatic oxidation of methane by the bacterium Methylococcus capsulatus].

The cytochrome c spectrally related to cco cytochromes has been isolated and purified from the methane-oxidizing bacterium Methylococcus capsulatus. The cytochrome binds CO but does not bind other substrates of methane monooxygenase, does not activate the methane monooxygenase reaction and is not a component of methane monooxygenase. In the methanol dehydrogenase enzymatic system cytochrome cco functions as electron acceptor. A possible role of cytochrome cco as electron carrier intermediate in the sequence of the dehydrogenase and oxidase enzymatic systems of M. capsulatus is discussed.

Carbon Monoxide↗
PubMed2001

Crystal structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath) demonstrating geometrical variability at the dinuclear iron active site.

The oxidation of methane to methanol is performed at carboxylate-bridged dinuclear iron centers in the soluble methane monooxygenase hydroxylase (MMOH). Previous structural studies of MMOH, and the related R2 subunit of ribonucleotide reductase, have demonstrated the occurrence of carboxylate shifts involving glutamate residues that ligate the catalytic iron atoms. These shifts are thought to have important mechanistic implications. Recent kinetic and theoretical studies have also emphasized the importance of hydrogen bonding and pH effects at the active site. We report here crystal structures of MMOH from Methylococcus capsulatus (Bath) in the diiron(II), diiron(III), and mixed-valent Fe(II)Fe(III) oxidation states, and at pH values of 6.2, 7.0, and 8.5. These structures were investigated in an effort to delineate the range of possible motions at the MMOH active site and to identify hydrogen-bonding interactions that may be important in understanding catalysis by the enzyme. Our results present the first view of the diiron center in the mixed-valent state, and they indicate an increased lability for ferrous ions in the enzyme. Alternate conformations of Asn214 near the active site according to redox state and a distortion in one of the alpha-helices adjacent to the metal center in the diiron(II) state have also been identified. These changes alter the surface of the protein in the vicinity of the catalytic core and may have implications for small-molecule accessibility to the active site and for protein component interactions in the methane monooxygenase system. Collectively, these results help to explain previous spectroscopic observations and provide new insight into catalysis by the enzyme.

Binding Sites↗
PubMed1990

Cloning, sequencing and expression of the glutamine synthetase structural gene (glnA) from the obligate methanotroph Methylococcus capsulatus (Bath).

The structural gene (glnA) encoding the ammonia-assimulation enzyme glutamine synthetase (GS) has been cloned from the obligate methanotroph Methylococcus capsulatus (Bath). Complementation of Escherichia coli glnA mutants was demonstrated. In vitro expression analysis revealed that the cloned glnA gene coded for a polypeptide of apparent Mr 60,000, as determined by PAGE. Expression of the M. capsulatus (Bath) glnA gene in E. coli was regulated by nitrogen levels in an Ntr+ but not an Ntr- background. The nucleotide sequence of the M. capsulatus (Bath) glnA gene and flanking sequences was determined. This gene, of 1407 bp, encoded a polypeptide of Mr 51717 containing 468 amino acids. The 5' leader region contained three putative promoters. Promoters P1 and P3 resembled the canonical -10 -35 E. coli-type promoter. Promoter P2, which was located between P1 and P3, resembled the NtrA-dependent promoters of enteric organisms. A potential NtrC-binding site was also determined, flanking the Pribnow box at P1. Comparisons of nucleotide-derived amino acid sequences of GS enzymes from prokaryotes and eukaryotes with GS from M. capsulatus are made.

Amino Acid Sequence↗
PubMed1991

Redox properties of the hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath). Effects of protein B, reductase, and substrate.

The reduction potentials of the hydroxylase component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been investigated through potentiometric titrations. The potentials were determined by EPR spectroscopic quantitation of the mixed valent hydroxylase as a function of added sodium dithionite in the presence of appropriate mediators. The reduction of the oxidized Fe(III).Fe(III) form to the mixed valent Fe(II).Fe(III) form occurs at 48 mV versus NHE while the potential for the formation of the fully reduced Fe(II).Fe(II) species from the mixed valent form was determined to be -135 mV. Addition of the substrate propylene to the hydroxylase did not have a major effect on the reduction potentials. Introduction of the protein B and the reductase components, however, completely inhibited reduction of the hydroxylase at potentials as far negative as -200 mV. Addition of propylene to all three methane monooxygenase components greatly facilitated hydroxylase reduction. Under these conditions, the fully reduced form of the protein was obtained at potentials of greater than 150 mV. This high redox potential indicates that the oxidized form of the protein is highly reactive, as required for methane oxidation. The present results reveal aspects of how both protein B and substrate can regulate electron transfer into and out of the hydroxylase component of methane monooxygenase.

Dithionite↗
PubMed1971

Pteridines produced by Methylococcus capsulatus. Isolation and identification of a neopterin 2':3'-phosphate.

Three pteridines have been isolated from the methane- or methanol-oxidizing bacterium Methylococcus capsulatus. Two of these are known compounds, 2-amino-6-carboxy-4-hydroxypteridine and 2-amino-4-hydroxy-6-methylpteridine. The third is shown by degradative and synthetic experiments to be l-threo-neopterin 2':3'-phosphate. Labelling experiments show that both the pteridine moiety and phosphate residue are derived from a single GTP molecule. The possible metabolic significance of these compounds in methanol oxidation is discussed.

Bacteria↗
PubMed1985

Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Copper(I), copper(II) and silver ions have been shown to be potent inhibitors of purified soluble methane monooxygenase (MMO) of Methylococcus capsulatus (Bath). A weaker inhibition has been observed with zinc and cadmium ions. Proteins A and B of soluble MMO are unaffected by copper but protein C is rapidly and irreversibly inhibited. The site of copper inhibition has been shown to be primarily at the iron-sulphur centre of protein C with a secondary effect at the FAD centre when the copper(II):protein C ratio is high. Copper appears to bring about the inhibition of soluble MMO by interacting with protein C to disrupt the protein structure causing, firstly, the loss of the iron-sulphur centre, preventing the transfer of electrons from protein C to protein A, and secondly, the loss of FAD preventing the protein from accepting electrons from NADH. Inhibition and spectral data are provided to support this thesis. The inactivation of protein C is associated with the tight binding of four Cu atoms to each protein C molecule. These data extend our knowledge of how copper, which is known to have a key role in the cellular location of MMO, interacts with and rapidly and irreversibly inactivates the soluble form of this enzyme.

Binding Sites↗
PubMed1997

Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by proteolysis can be overcome by a Gly to Gln modification.

The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full-length active form and truncated forms, B' and B". Electrospray ionisation mass spectrometry (ESI-MS) was used to identify a cleavage site between Met12 and Gly13, such that 12 amino acids were lost from the N-terminus of protein B. This truncate was designated B' and molecular masses were assigned to proteins B and B' of 15,852.6+/-0.4 Da and 14,629.5+/-0.3 Da, respectively. A cleavage site between Gln29 and Val30 was also identified such that 29 amino acids were lost from the N-terminus of protein B. This truncate was designated B" and had a molecular mass of 12,709.93+/-0.02 Da. Proteins B' and B" were found to be inactive in the sMMO system. Addition of protease inhibitors or the heterologous expression of protein B in various strains of lon-deficient or ompT-deficient Escherichia coli, did not inhibit B' formation. Expression of protein B as a glutathione S-transferase fusion protein and subsequent purification of protein B from E. coli using affinity chromatography resulted in preparations of protein B with higher enzyme activities than that of wild-type protein B. However, ESI-MS confirmed that protein B' was still present. Alteration of the Met12-Gly13 cleavage site to Met12-Gln13 revealed that the stability of G13Q at 20 degrees C and 37 degrees C was higher than that of wild-type preparations. ESI-MS indicated that protein B' was absent and could only be identified after prolonged incubation at room temperature. The amount of active protein B present in the cell may be controlled by protein B cleavage, thereby regulating electron transfer. Alternatively, it may allow protein B to maintain a certain conformation necessary for enzyme activity and this may control the activity of sMMO in response to the supply of methane to the cell.

Amino Acid Sequence↗
PubMed1993

Activation of the hydroxylase of sMMO from Methylococcus capsulatus (Bath) by hydrogen peroxide.

Hydrogen peroxide can activate the non-heme binuclear iron-containing hydroxylase of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) in the catalysis of oxidation of methane and other sMMO substrates. The reductase, protein B, O2 and NADH are normally required for catalytic activity, but can be replaced by H2O2 serving as the source of both oxygen and electrons for the reaction. Similar results have been observed in a different strain of MMO from Methylosinus trichosporium OB3b (Andersson, K.K., Froland, W.A., Lee, S.-K. and Lipscomb, J.D. (1991) New J. Chem. 15, 410-415). The Km,app for H2O2 was found to be 66 mM. Labelled oxygen experiments show that the oxygen atom in the product in the peroxide driven system is derived from H2O2 and not O2. Using C2-C5 alkanes and 2-butene as substrates it was shown that the product distribution differed in the complete sMMO and H2O2-driven systems, indicating that more than one pathway is available to the enzyme. Protein B, which is required for catalytic activity in the complete system, was found to be an inhibitor of the hydroxylase/H2O2 system. It was also observed that protein B not only affected the activity, but also the selectivity of carbon hydroxylation with 2-methylbutane as substrate.

Dose-Response Relationship, Drug↗
PubMed1998

The hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) exists in several forms as shown by electrospray-ionisation mass spectrometry.

The hydroxylase of the soluble methane monooxygenase from the bacterium Methylococcus capsulatus (Bath) has been investigated by means of electrospray-ionisation mass spectrometry (ESI-MS) and liquid chromatography ESI-MS (LC/ESI-MS). The hydroxylase is a non-heme diiron protein consisting of three pairs of non-identical subunits (alpha approximately 60 kDa, beta approximately 45 kDa and gamma approximately 20 kDa). Liquid chromatographic separation of the hydroxylase subunits was required before MS analysis in order to detect the alpha-subunit. The masses measured for the three subunits were found to disagree with those calculated from their gene sequences. Experiments involving the use of CNBr and trypsin cleavage followed by LC/ESI-MS and MS/MS analyses permitted the location and correction of errors in the sequences deduced from the use of cDNA. The ESI-MS results also showed that the alpha-subunit of the hydroxylase exists in multiple forms which result from cleavage of the protein. This observation explains a number of enigmatic features of the protein previously reported in the literature and illustrates the pivotal role of ESI-MS in complementing data obtained from molecular biology for the characterisation of the primary sequence of proteins.

Cyanogen Bromide↗
PubMed1973

Physiological studies of methane- and methanol-oxidizing bacteria: immunological comparison of a primary alcohol dehydrogenase from Methylococcus capsulatus and Pseudomonas sp. M27.

A primary alcohol dehydrogenase was purified from cell extracts of two apparently unrelated microorganisms, namely, Pseudomonas sp. M27 and Methylococcus capsulatus. Rabbit antiserum prepared against the purified enzyme from M. capsulatus revealed distinctive antigenic determinants by quantitative and gel precipitin reactions. Rabbit antiserum to M27 enzyme detected both distinctive and shared antigenic determinants. Certain methane- and methanol-oxidizing bacteria were grouped on the basis of serological cross-reacting enzyme specificities.

Alcohol Oxidoreductases↗
PubMed1999

High-molecular-mass multi-c-heme cytochromes from Methylococcus capsulatus bath.

The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6. 0. The heme c concentration was estimated to be 8.2 +/- 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118, 620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94, 000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome.

Amino Acid Sequence↗
PubMed1977

The soluble methane mono-oxygenase of Methylococcus capsulatus (Bath). Its ability to oxygenate n-alkanes, n-alkenes, ethers, and alicyclic, aromatic and heterocyclic compounds.

1. Methane mono-oxygenase of Methylococcus capsulatus (Bath) catalyses the oxidation of various substituted methane derivatives including methanol. 2. It is a very non-specific oxygenase and, in some of its catalytic properties, apparently resembles the analogous enzyme from Methylomonas methanica but differs from those found in Methylosinus trichosporium and Methylomonas albus. 3. CO is oxidized to CO2. 4. C1-C8 n-alkanes are hydroxylated, yielding mixtures of the corresponding 1- and 2-alcohols; no 3- or 4-alcohols are formed. 5. Terminal alkenes yield the corresponding 1,2-epoxides. cis- or trans-but-2-ene are each oxidized to a mixture of 2,3-epoxybutane and but-2-en-1-ol with retention of the cis or trans configuration in both products; 2-butanone is also formed from cis-but-2-ene only. 6. Dimethyl ether is oxidized. Diethyl ether undergoes sub-terminal oxidation, yielding ethanol and ethanal in equimolar amounts. 7. Methane mono-oxygenase also hydroxylates cyclic alkanes and aromatic compounds. However, styrene yields only styrene epoxide and pyridine yields only pyridine N-oxide. 8. Of those compounds tested, only NADPH can replace NADH as electron donor.

Alkanes↗
PubMed1975

Oxidation of C1 Compounds by Particulate fractions from Methylococcus capsulatus: distribution and properties of methane-dependent reduced nicotinamide adenine dinucleotide oxidase (methane hydroxylase).

Cell-free particulate fractions of extracts from the obligate methylotroph Methylococcus capsulatus catalyze the reduced nicotinamide adenine dinucleotide (NADH) and O2-dependent oxidation of methane (methane hydroxylase). The only oxidation product detected was formate. These preparations also catalyze the oxidation of methanol and formaldehyde to formate in the presence or absence of phenazine methosulphate with oxygen as the terminal electron acceptor. Methane hydroxylase activity cannot be reproducibly obtained from disintegrated cell suspensions even though the whole cells actively respired when methane was presented as a substrate. Varying the disintegration method or extraction medium had no significant effect on the activities obtained. When active particles were obtained, hydroxylase activity was stable at 0 C for days. Methane hydroxylase assays were made by measuring the methane-dependent oxidation of NADH by O2. In separate experiments, methane consumption and the accumulation of formate were also demonstrated. Formate is not oxidized by these particulate fractions. The effects of particle concentration, temperature, pH, and phosphate concentration on enzymic activity are described. Ethane is utilized in the presence of NADH and O2. The stoichiometric relationships of the reaction(s) with methane as substrate were not established since (i) the presumed initial product, methanol, is also oxidized to formate, and (ii) the contribution that NADH oxidase activity makes to the observed consumption of reactants could not be assessed in the presence of methane. Studies with known inhibitors of electron transport systems indicate that the path of electron flow from NADH to oxygen is different for the NADH oxidase, methane hydroxylase, and methanol oxidase activities.

Bacteria↗