The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Stephen J Lippard - One of the best experts on this subject based on the ideXlab platform.
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Methane Monooxygenase: Functionalizing Methane at Iron and Copper
Metal ions in life sciences, 2014Co-Authors: Matthew H. Sazinsky, Stephen J LippardAbstract:Methane Monooxygenases (MMOs) catalyze the conversion of Methane to methanol as the first committed step in the assimilation of this hydrocarbon into biomass and energy by methanotrophs, thus playing a significant role in the biogeochemistry of this potent greenhouse gas. Two distinct enzymes, a copper-dependent membrane protein, particulate Methane Monooxygenase (pMMO), and an iron-dependent cytosolic protein, soluble Methane Monooxygenase (sMMO), carry out this transformation using large protein scaffolds that help to facilitate the timely transport of hydrocarbon, O2, proton, and electron substrates to buried dimetallic active sites. For both enzymes, reaction of the reduced metal centers with O2 leads to intermediates that activate the relatively inert C–H bonds of hydrocarbons to yield oxidized products. Among synthetic and biological catalysts, MMOs are unique because they are the only ones known to hydroxylate Methane at ambient temperatures. As a need for new industrial catalysts and green chemical transformations increases, understanding how the different MMO metal centers efficiently accomplish this challenging chemistry has become the focus of intense study. This chapter examines current understanding of the sMMO and pMMO protein structures, their methods for substrate channeling, and mechanisms for the dimetallic activation of O2 and C–H bonds.
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electron transfer control in soluble Methane Monooxygenase
Journal of the American Chemical Society, 2014Co-Authors: Weixue Wang, Roxana E Iacob, Rebecca P Luoh, John R Engen, Stephen J LippardAbstract:The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent Monooxygenases (BMMs) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determine the reductase binding site on the hydroxylase of a BMM enzyme, soluble Methane Monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism.
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dioxygen activation in soluble Methane Monooxygenase
Accounts of Chemical Research, 2011Co-Authors: Christine E Tinberg, Stephen J LippardAbstract:The controlled oxidation of Methane to methanol is a chemical transformation of great value, particularly in the pursuit of alternative fuels, but the reaction remains underutilized industrially because of inefficient and costly synthetic procedures. In contrast, Methane Monooxygenase enzymes (MMOs) from methanotrophic bacteria achieve this chemistry efficiently under ambient conditions. In this Account, we discuss the first observable step in the oxidation of Methane at the carboxylate-bridged diiron active site of the soluble MMO (sMMO), namely, the reductive activation of atmospheric O2. The results provide benchmarks against which the dioxygen activation mechanisms of other bacterial multicomponent Monooxygenases can be measured.Molecular oxygen reacts rapidly with the reduced diiron(II) cen-ter of the hydroxylase component of sMMO (MMOH). The first spectroscopically characterized intermediate that results from this process is a peroxodiiron(III) species, P*, in which the iron atoms have identical env...
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Dioxygen activation in Methane Monooxygenase: a theoretical study.
Journal of the American Chemical Society, 2004Co-Authors: Benjamin F. Gherman, Stephen J Lippard, Mu-hyun Baik, Richard A. FriesnerAbstract:Using broken-symmetry unrestricted Density Functional Theory, the mechanism of enzymatic dioxygen activation by the hydroxylase component of soluble Methane Monooxygenase (MMOH) is determined to atomic detail. After a thorough examination of mechanistic alternatives, an optimal pathway was identified. The diiron(II) state Hred reacts with dioxygen to give a ferromagnetically coupled diiron(II,III) Hsuperoxo structure, which undergoes intersystem crossing to the antiferromagnetic surface and affords Hperoxo, a symmetric diiron(III) unit with a nonplanar μ-η2:η2-O22- binding mode. Homolytic cleavage of the O−O bond yields the catalytically competent intermediate Q, which has a di (μ-oxo)diiron(IV) core. A carboxylate shift involving Glu243 is essential to the formation of the symmetric Hperoxo and Q structures. Both thermodynamic and kinetic features agree well with experimental data, and computed spin-exchange coupling constants are in accord with spectroscopic values. Evidence is presented for pH-independ...
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Mechanistic studies on the hydroxylation of Methane by Methane Monooxygenase.
Chemical reviews, 2003Co-Authors: Mu-hyun Baik, Richard A. Friesner, Martin Newcomb, Stephen J LippardAbstract:Methanotrophs are bacteria that live on Methane as their only source of carbon.1 The first step in their utilization of this simplest of all hydrocarbons is its selective conversion to methanol. Subsequent biochemical pathways transform methanol to formaldehyde, which in turn is processed into biomass. Further oxidation of formaldehyde to carbon dioxide provides energy that is stored for later use as NADH.2 The conversion of Methane to methanol is catalyzed at the active site of a metalloenzyme known as Methane Monooxygenase, or MMO.3-9
J. Colin Murrell - One of the best experts on this subject based on the ideXlab platform.
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Involvement of MmoR and MmoG in the transcriptional activation of soluble Methane Monooxygenase genes in Methylosinus trichosporium OB3b
FEMS microbiology letters, 2009Co-Authors: Julie Scanlan, Marc G. Dumont, J. Colin MurrellAbstract:Methanotrophs oxidize Methane to methanol using the enzyme Methane Monooxygenase. Methylosinus trichosporium OB3b has two such enzymes: a membrane-bound particulate Methane Monooxygenase (pMMO) and a soluble, cytoplasmic Methane Monooxygenase (sMMO). In methanotrophs possessing both enzymes, the expression of the genes encoding sMMO and pMMO is regulated by copper ions, with sMMO expressed solely when copper is limiting. Virtually nothing is known about the specific machinery involved in the copper-regulated transcription of mmo genes except the identification of two proteins necessary for the expression: a sigma(54)-dependent transcriptional activator, MmoR, and a putative GroEL-like chaperone, MmoG. Genes encoding mmoR and mmoG are located immediately upstream of those encoding sMMO in the genome of M. trichosporium OB3b. Here, we use a green fluorescent protein promoter probe vector to show that nearly the complete intergenic DNA sequence between mmoG and mmoX is absolutely required for transcriptional activation. Furthermore, we used gel-shift assays to demonstrate that both MmoR and MmoG were required for protein binding to this region of DNA.
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Molecular biology and regulation of Methane Monooxygenase
Archives of Microbiology, 2000Co-Authors: J. Colin Murrell, Bettina Gilbert, Ian R McdonaldAbstract:Methanotrophs are ubiquitous in the environment and play an important role in mitigating global warming due to Methane. They are also potentially interesting for industrial applications such as production of bulk chemicals or bioremediation. The first step in the oxidation of Methane is the conversion to methanol by Methane Monooxygenase, the key enzyme, which exists in two forms: the cytoplasmic, soluble Methane Monooxygenase (sMMO) and the membrane-bound, particulate Methane Monooxygenase (pMMO). This paper reviews the biochemistry and molecular biology of both forms of MMO. In the past few years there have been many exciting new findings. sMMO components have been expressed in heterologous and homologous hosts. The pMMO has been purified and biochemically studied in some detail and the genes encoding the pMMO have been sequenced. Copper ions have been shown to play a key role in regulating the expression of both MMO enzyme complexes. We also present a model for copper regulation based on results from Northern analysis, primer-extensions and new sequence data, and raise a number of unanswered questions for future studies.
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The particulate Methane Monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs
FEMS microbiology letters, 1997Co-Authors: Ian R Mcdonald, J. Colin MurrellAbstract:The particulate Methane Monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate Methane Monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using Methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.
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Evidence that participate Methane Monooxygenase and ammonia Monooxygenase may be evolutionarily related
Fems Microbiology Letters, 1995Co-Authors: Andrew J. Holmes, Andria M. Costello, Mary E. Lidstrom, J. Colin MurrellAbstract:Abstract Genes encoding paniculate Methane Monooxygenase and ammonia Monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate Methane Monooxygenase and ammonia Monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides ( pmoA and amoA ) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs ( α- and γ-Proteobacteria) and ammonia-oxidizing nitrifying bacteria ( β-and y-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the γ-Proteobacteria than to AmoA sequences. These results suggest that the particulate Methane Monooxygenase and ammonia Monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.
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Trichloroethylene oxidation by the membrane-associated Methane Monooxygenase in type I, type II and type X methanotrophs
Biodegradation, 1992Co-Authors: Alan A. Dispirito, J. Colin Murrell, Mary E. Lidstrom, Andrew K. Shiemke, Jay Gulledge, Cinder L. KremaAbstract: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.
Howard Dalton - One of the best experts on this subject based on the ideXlab platform.
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heterologous expression of soluble Methane Monooxygenase genes in methanotrophs containing only particulate Methane Monooxygenase
Archives of Microbiology, 1999Co-Authors: John S Lloyd, Howard Dalton, Paolo De Marco, J. C. MurrellAbstract:The methanotrophs Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b contain particulate Methane Monooxygenase (pMMO) and soluble Methane Monooxygenase (sMMO) genes. Other methanotrophs such as Methylomicrobium album BG8 and Methylocystis parvus OBBP contain only pMMO genes. Although molecular genetic techniques are poorly developed in methanotrophs, sMMO genes were expressed in methanotrophs normally containing only pMMO genes. This was achieved by conjugation using broad-host-range plasmids containing the native promoter and sMMO genes from Mc. capsulatus (Bath) and Ms. trichosporium OB3b. sMMO genes derived from Ms. trichosporium OB3b were expressed in an active form in Mcy. parvus OBBP and in Mm. album BG8. Therefore, all of the genes required for active sMMO synthesis were contained on the broad-host-range plasmids and were expressed in the heterologous hosts. Constitutive synthesis of pMMO was observed in Mm. album BG8 when grown at high and low copper-to-biomass ratios, while transcription of the recombinant sMMO genes was only observed under growth conditions of low copper-to-biomass ratios. Therefore, the regulatory protein(s) for sMMO synthesis was also present on the plasmid used, or the heterologous host contained a regulatory system for sMMO. Expression of sMMO genes in methanotrophs containing only pMMO will assist further investigations on the expression and regulation of MMO genes in methanotrophs.
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further evidence for multiple pathways in soluble Methane Monooxygenase catalysed oxidations from the measurement of deuterium kinetic isotope effects
FEBS Journal, 1994Co-Authors: Patricia C Wilkins, Howard Dalton, Christopher J Samuel, Jeffrey GreenAbstract: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, 7. A mechanistic pathway in which a substrate radical is formed from hydrogen atom abstraction by a ferryl species is believed to operate for CH4, the toluene –CH3 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 Methane-Monooxygenase-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.
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Further Evidence for Multiple Pathways in Soluble Methane‐Monooxygenase‐Catalysed Oxidations from the Measurement of Deuterium Kinetic Isotope Effects
European journal of biochemistry, 1994Co-Authors: Patricia C Wilkins, Howard Dalton, Christopher J Samuel, Jeffrey GreenAbstract: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, 7. A mechanistic pathway in which a substrate radical is formed from hydrogen atom abstraction by a ferryl species is believed to operate for CH4, the toluene –CH3 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 Methane-Monooxygenase-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.
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Evidence for two histidine ligands at the diiron site of Methane Monooxygenase.
European journal of biochemistry, 1992Co-Authors: Douglas Robert Drummond, S. Smith, Howard DaltonAbstract:Circular dichroism spectroscopy has shown the hydroxylase component of Methane Monooxygenase to have a high helical content. The apoprotein has the same secondary structure as the holoenzyme. Chemical modification shows 12 histidines to be reactive with diethylpyrocarbonate in the holoenzyme, whereas 14 are reactive in the apoenzyme. Two histidine residues are implicated as iron ligands. Further chemical modification results suggest a cysteine residue is in close proximity to the diiron centre.
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purification and characterisation of the soluble Methane Monooxygenase from methylosinus sporium 5 demonstrates the highly conserved nature of this enzyme in methanotrophs
Fems Microbiology Letters, 1991Co-Authors: S.j. Pilkington, Howard DaltonAbstract:The type II obligate methanotroph Methylosinus sporium 5 was shown to have the ability to produce either a soluble or particulate Methane Monooxygenase dependent on the copper to biomass ratio during growth. Two proteins of the soluble Methane Monooxygenase enzyme, proteins A (the hydroxylase) and C (the NADH-acceptor reductase) were purified and characterised, and shown to be very similar to those previously described in other organisms. Evidence is also presented for the existence of the third protein, protein B, in this enzyme complex.
Amy C. Rosenzweig - One of the best experts on this subject based on the ideXlab platform.
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Towards a unified understanding of the copper sites in particulate Methane Monooxygenase: an X-ray absorption spectroscopic investigation
Chemical Science, 2021Co-Authors: George E. Cutsail, Amy C. Rosenzweig, Matthew O Ross, Serena DebeerAbstract:Extended X-ray absorption fine structure spectroscopic analysis of particulate Methane Monooxygenase reveals only monocopper sites and investigates the possible origins of the previous observed dicopper signals.
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particulate Methane Monooxygenase contains only mononuclear copper centers
Science, 2019Co-Authors: Matthew O Ross, Amy C. Rosenzweig, Fraser Macmillan, Jingzhou Wang, Alex Nisthal, Thomas J Lawton, Barry D Olafson, Stephen L Mayo, Brian M HoffmanAbstract:Bacteria that oxidize Methane to methanol are central to mitigating emissions of Methane, a potent greenhouse gas. The nature of the copper active site in the primary metabolic enzyme of these bacteria, particulate Methane Monooxygenase (pMMO), has been controversial owing to seemingly contradictory biochemical, spectroscopic, and crystallographic results. We present biochemical and electron paramagnetic resonance spectroscopic characterization most consistent with two monocopper sites within pMMO: one in the soluble PmoB subunit at the previously assigned active site (CuB) and one ~2 nanometers away in the membrane-bound PmoC subunit (CuC). On the basis of these results, we propose that a monocopper site is able to catalyze Methane oxidation in pMMO.
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Architecture and active site of particulate Methane Monooxygenase.
Critical reviews in biochemistry and molecular biology, 2012Co-Authors: Megen A. Culpepper, Amy C. RosenzweigAbstract: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 frag...
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Metal reconstitution of particulate Methane Monooxygenase and heterologous expression of the pmoB subunit.
Methods in enzymology, 2011Co-Authors: Stephen M. Smith, Ramakrishnan Balasubramanian, Amy C. RosenzweigAbstract:Particulate Methane Monooxygenase (pMMO) is a multisubunit metalloenzyme complex used by methanotrophic bacteria to oxidize Methane in the first step of carbon assimilation and energy production. In this chapter, we detail methods to prepare metal free (apo) membrane-bound pMMO and to reconstitute apo pMMO with metal ions. We also describe protocols to clone, express, and refold metal-loaded soluble domain constructs of the pmoB subunit. These approaches were used to address fundamental questions concerning the metal content and location of the pMMO active site.
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Handbook of Metalloproteins - Particulate Methane Monooxygenase
Handbook of Metalloproteins, 2008Co-Authors: Amy C. RosenzweigAbstract: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 CuCu interaction at 2.5 A that increases to 2.6 A 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. 3D Structure Keywords: Methane Monooxygenase; pMMO; copper; Methane oxidation; membrane protein; hydroxylase; methanotroph
Cinder L. Krema - One of the best experts on this subject based on the ideXlab platform.
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differential inhibition in vivo of ammonia Monooxygenase soluble Methane Monooxygenase and membrane associated Methane Monooxygenase by phenylacetylene
Environmental Microbiology, 2000Co-Authors: Sonny Lontoh, Alan B Hooper, Alan A. Dispirito, Cinder L. Krema, Mark Whittaker, Jeremy D. SemrauAbstract:Phenylacetylene was investigated as a differential inhibitor of ammonia Monooxygenase (AMO), soluble Methane Monooxygenase (sMMO) and membrane-associated or particulate Methane Monooxygenase (pMMO) in vivo. At phenylacetylene concentrations > 1 microM, whole-cell AMO activity in Nitrosomonas europaea was completely inhibited. Phenylacetylene concentrations above 100 microM inhibited more than 90% of sMMO activity in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b. In contrast, activity of pMMO in M. trichosporium OB3b, M. capsulatus Bath, Methylomicrobium album BG8, Methylobacter marinus A45 and Methylomonas strain MN was still measurable at phenylacetylene concentrations up to 1,000 microM. AMO of Nitrosococcus oceanus has more sequence similarity to pMMO than to AMO of N. europaea. Correspondingly, AMO in N. oceanus was also measurable in the presence of 1,000 microM phenylacetylene. Measurement of oxygen uptake indicated that phenylacetylene acted as a specific and mechanistic-based inhibitor of whole-cell sMMO activity; inactivation of sMMO was irreversible, time dependent, first order and required catalytic turnover. Corresponding measurement of oxygen uptake in whole cells of methanotrophs expressing pMMO showed that pMMO activity was inhibited by phenylacetylene, but only if Methane was already being oxidized, and then only at much higher concentrations of phenylacetylene and at lower rates compared with sMMO. As phenylacetylene has a high solubility and low volatility, it may prove to be useful for monitoring methanotrophic and nitrifying activity as well as identifying the form of MMO predominantly expressed in situ.
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Differential inhibition in vivo of ammonia Monooxygenase, soluble Methane Monooxygenase and membrane‐associated Methane Monooxygenase by phenylacetylene
Environmental microbiology, 2000Co-Authors: Sonny Lontoh, Alan B Hooper, Alan A. Dispirito, Cinder L. Krema, Mark Whittaker, Jeremy D. SemrauAbstract:Phenylacetylene was investigated as a differential inhibitor of ammonia Monooxygenase (AMO), soluble Methane Monooxygenase (sMMO) and membrane-associated or particulate Methane Monooxygenase (pMMO) in vivo. At phenylacetylene concentrations > 1 microM, whole-cell AMO activity in Nitrosomonas europaea was completely inhibited. Phenylacetylene concentrations above 100 microM inhibited more than 90% of sMMO activity in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b. In contrast, activity of pMMO in M. trichosporium OB3b, M. capsulatus Bath, Methylomicrobium album BG8, Methylobacter marinus A45 and Methylomonas strain MN was still measurable at phenylacetylene concentrations up to 1,000 microM. AMO of Nitrosococcus oceanus has more sequence similarity to pMMO than to AMO of N. europaea. Correspondingly, AMO in N. oceanus was also measurable in the presence of 1,000 microM phenylacetylene. Measurement of oxygen uptake indicated that phenylacetylene acted as a specific and mechanistic-based inhibitor of whole-cell sMMO activity; inactivation of sMMO was irreversible, time dependent, first order and required catalytic turnover. Corresponding measurement of oxygen uptake in whole cells of methanotrophs expressing pMMO showed that pMMO activity was inhibited by phenylacetylene, but only if Methane was already being oxidized, and then only at much higher concentrations of phenylacetylene and at lower rates compared with sMMO. As phenylacetylene has a high solubility and low volatility, it may prove to be useful for monitoring methanotrophic and nitrifying activity as well as identifying the form of MMO predominantly expressed in situ.
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Trichloroethylene oxidation by the membrane-associated Methane Monooxygenase in type I, type II and type X methanotrophs
Biodegradation, 1992Co-Authors: Alan A. Dispirito, J. Colin Murrell, Mary E. Lidstrom, Andrew K. Shiemke, Jay Gulledge, Cinder L. KremaAbstract: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.
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Trichloroethylene oxidation by the membrane-associated Methane Monooxygenase in type I, type II and type X methanotrophs
Biodegradation, 1991Co-Authors: Alan A. Dispirito, J. Colin Murrell, Mary E. Lidstrom, Andrew K. Shiemke, Jay Gulledge, Cinder L. KremaAbstract: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.