RESUMEN
A family of bacterial copper storage proteins (the Csps) possess thiolate-lined four-helix bundles whose cores can be filled with Cu(I) ions. The majority of Csps are cytosolic (Csp3s), and in vitro studies carried out to date indicate that the Csp3s from Methylosinus trichosporium OB3b (MtCsp3), Bacillus subtilis (BsCsp3), and Streptomyces lividans (SlCsp3) are alike. Bioinformatics have highlighted homologues with potentially different Cu(I)-binding properties from these characterized "classical" Csp3s. Determination herein of the crystal structure of the protein (RkCsp3) from the methanotroph Methylocystis sp. strain Rockwell with Cu(I) bound identifies this as the first studied example of a new subgroup of Csp3s. The most significant structural difference from classical Csp3s is the presence of only two Cu(I) sites at the mouth of the bundle via which Cu(I) ions enter and leave. This is due to the absence of three Cys residues and a His-containing motif, which allow classical Csp3s to bind five to six Cu(I) ions in this region. Regardless, RkCsp3 exhibits rapid Cu(I) binding and the fastest measured Cu(I) removal rate for a Csp3 when using high-affinity ligands as surrogate partners. New experiments on classical Csp3s demonstrate that their His-containing motif is not essential for fast Cu(I) uptake and removal. Other structural features that could be important for these functionally relevant in vitro properties are discussed.
Asunto(s)
Proteínas Bacterianas , Methylosinus trichosporium , Proteínas Bacterianas/química , Cobre/química , Methylosinus trichosporium/química , Methylosinus trichosporium/metabolismoRESUMEN
Aerobic methane oxidation (MOx) depends critically on the availability of copper (Cu) as a crucial component of the metal centre of particulate methane monooxygenase, one of the main enzymes involved in MOx. Some methanotrophs have developed Cu acquisition strategies, in which they exude Cu-binding ligands termed chalkophores under conditions of low Cu availability. A well-characterised chalkophore is methanobactin (mb), exuded by the microaerophilic methanotroph Methylosinus trichosporium OB3b. Aerobic methanotrophs generally reside close to environmental oxic-anoxic interfaces, where the formation of Cu sulphide phases can aggravate the limitation of bioavailable Cu due to their low solubility. The reactivity of chalkophores towards such Cu sulphide mineral phases has not yet been investigated. In this study, a combination of dissolution experiments and equilibrium modelling was used to examine the dissolution and solubility of bulk and nanoparticulate Cu sulphide minerals in the presence of mb as influenced by pH, oxygen and natural organic matter. In general, we show that mb is effective at increasing the dissolved Cu concentrations in the presence of a variety of Cu sulphide phases that may potentially limit Cu bioavailability. More Cu was mobilised per mole of mb from Cu sulphide nanoparticles compared with well-crystalline bulk covellite (CuS). In general, the efficacy of mb at mobilising Cu from Cu sulphides is pH-dependent. At lower pH, e.g. pH 5, mb was ineffective at solubilizing Cu. The presence of mb increased dissolved Cu concentrations between pH 7 and 8.5, where the solubility of all Cu sulphides is generally low, both in the presence and absence of oxygen. These results suggest that chalkophore-promoted Cu mobilisation from sulphide phases is an effective extracellular mechanism for increasing dissolved Cu concentrations at oxic-anoxic interfaces, particularly in the neutral to slightly alkaline pH range. This suggests that aerobic methanotrophs may be able to fulfil their Cu requirements via the exudation of mb in natural environments where the bioavailability of Cu is constrained by very stable Cu sulphide phases.
Asunto(s)
Cobre , Methylosinus trichosporium , Cobre/química , Concentración de Iones de Hidrógeno , Imidazoles , Methylosinus trichosporium/química , Minerales , Oligopéptidos , Oxígeno , SulfurosRESUMEN
Full activity of soluble methane monooxygenase (sMMO) depends upon the formation of a 1:1 complex of the regulatory protein MMOB with each alpha subunit of the (αßγ)2 hydroxylase, sMMOH. Previous studies have shown that mutations in the core region of MMOB and in the N- and C-termini cause dramatic changes in the rate constants for steps in the sMMOH reaction cycle. Here, X-ray crystal structures are reported for the sMMOH complex with two double variants within the core region of MMOB, DBL1 (N107G/S110A), and DBL2 (S109A/T111A), as well as two variants in the MMOB N-terminal region, H33A and H5A. DBL1 causes a 150-fold decrease in the formation rate constant of the reaction cycle intermediate P, whereas DBL2 accelerates the reaction of the dinuclear Fe(IV) intermediate Q with substrates larger than methane by three- to fourfold. H33A also greatly slows P formation, while H5A modestly slows both formation of Q and its reactions with substrates. Complexation with DBL1 or H33A alters the position of sMMOH residue R245, which is part of a conserved hydrogen-bonding network encompassing the active site diiron cluster where P is formed. Accordingly, electron paramagnetic resonance spectra of sMMOH:DBL1 and sMMOH:H33A complexes differ markedly from that of sMMOH:MMOB, showing an altered electronic environment. In the sMMOH:DBL2 complex, the position of M247 in sMMOH is altered such that it enlarges a molecular tunnel associated with substrate entry into the active site. The H5A variant causes only subtle structural changes despite its kinetic effects, emphasizing the precise alignment of sMMOH and MMOB required for efficient catalysis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Methylosinus trichosporium/metabolismo , Oxigenasas/metabolismo , Proteínas Bacterianas/química , Cristalografía por Rayos X , Methylosinus trichosporium/química , Modelos Moleculares , Oxigenasas/química , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a CuI primary-sphere ligand, being positioned â¼2.9 Å away from the CuI ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis.
Asunto(s)
Proteínas Bacterianas/química , Cobre/química , Quinurenina/química , Metaloproteínas/química , Methylosinus trichosporium/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Cristalografía por Rayos X , Quinurenina/biosíntesis , Quinurenina/genética , Metaloproteínas/genética , Metaloproteínas/metabolismo , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Dominios ProteicosRESUMEN
Methanotrophs synthesize methanobactin, a secondary metabolite that binds copper with an unprecedentedly high affinity. Such a strategy may provide methanotrophs a "copper monopoly" that can inhibit the activity of copper-containing enzymes of other microbes, e.g., copper-dependent N2O reductases. Here, we show that methanobactin from Methylosinus trichosporium OB3b inhibited N2O reduction in denitrifiers. When Pseudomonas stutzeri DCP-Ps1 was incubated in cocultures with M. trichosporium OB3b or with purified methanobactin from M. trichosporium OB3b, stoichiometric N2O production was observed from NO3- reduction, whereas no significant N2O accumulation was observed in cocultures with a mutant defective in methanobactin production. Copper uptake by P. stutzeri DCP-Ps1 was inhibited by the presence of purified methanobactin, leading to a significant downregulation of nosZ transcription. Similar findings were observed with three other denitrifier strains. These results suggest that in situ stimulation of methanotrophs can inadvertently increase N2O emissions, with the potential for increasing net greenhouse gas emissions.
Asunto(s)
Imidazoles/farmacología , Methylosinus trichosporium/metabolismo , Óxido Nitroso/metabolismo , Oligopéptidos/farmacología , Pseudomonas stutzeri/efectos de los fármacos , Transporte Biológico , Cobre/metabolismo , Imidazoles/metabolismo , Methylosinus trichosporium/química , Methylosinus trichosporium/genética , Oligopéptidos/metabolismo , Oxidación-Reducción , Pseudomonas stutzeri/metabolismoRESUMEN
Kα high-energy-resolution fluorescence detected X-ray absorption spectroscopy (HERFD XAS) provides a powerful tool for overcoming the limitations of conventional XAS to identify the electronic structure and coordination environment of metalloprotein active sites. Herein, Fe Kα HERFD XAS is applied to the diiron active site of soluble methane monooxygenase (sMMO) and to a series of high-valent diiron model complexes, including diamond-core [FeIV2(µ-O)2(L)2](ClO4)4] (3) and open-core [(OâFeIV-O-FeIV(OH)(L)2](ClO4)3 (4) models (where, L = tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) (TPA*)). Pronounced differences in the HERFD XAS pre-edge energies and intensities are observed for the open versus closed Fe2O2 cores in the model compounds. These differences are reproduced by time-dependent density functional theory (TDDFT) calculations and allow for the pre-edge energies and intensity to be directly correlated with the local active site geometric and electronic structure. A comparison of the model complex HERFD XAS data to that of MMOHQ (the key intermediate in methane oxidation) is supportive of an open-core structure. Specifically, the large pre-edge area observed for MMOHQ may be rationalized by invoking an open-core structure with a terminal FeIVâO motif, though further modulations of the core structure due to the protein environment cannot be ruled out. The present study thus motivates the need for additional experimental and theoretical studies to unambiguously assess the active site conformation of MMOHQ.
Asunto(s)
Fluorescencia , Oxigenasas/química , Oxigenasas/metabolismo , Espectroscopía de Absorción de Rayos X , Biocatálisis , Peróxido de Hidrógeno/química , Hierro/química , Methylosinus trichosporium/química , Methylosinus trichosporium/enzimología , Oxidación-Reducción , SolubilidadRESUMEN
RATIONALE: Traditional investigation of bacteriohopanepolyols (BHPs) has relied on derivatisation by acetylation prior to gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/MS (LC/MS) analysis. Here, modern chromatographic techniques (ultrahigh-performance liquid chromatography (UPLC)) and new column chemistries were tested to develop a method for BHP analysis without the need for derivatisation. METHODS: Bacterial culture and sedimentary lipid extracts were analysed using a Waters Acquity Xevo TQ-S triple quadrupole mass spectrometer in positive ion atmospheric pressure chemical ionisation (APCI) mode. Waters BEH C18 and ACE Excel C18 were the central columns evaluated using a binary solvent gradient with 0.1% formic acid in the polar solvent phase in order to optimise performance and selectivity. RESULTS: Non-amine BHPs and adenosylhopane showed similar performance on each C18 column; however, BHP-containing terminal amines were only identified eluting from the ultra-inert ACE Excel C18 column. APCI-MS/MS product ion scans revealed significant differences in fragmentation pathways from previous methods for acetylated compounds. The product ions used for targeted multiple reaction monitoring (MRM) are summarised. CONCLUSIONS: UPLC/MS/MS analysis using an ACE Excel C18 column produced superior separation for amine-containing BHPs and reduced run times from 60 to 9 min compared with previous methods. Unexpected variations in fragmentation pathways between structural subgroups must be taken into account when optimising MRM transitions for future quantitative studies. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Lípidos de la Membrana/química , Methylococcus capsulatus/química , Methylosinus trichosporium/química , Espectrometría de Masas en Tándem/métodos , Lípidos de la Membrana/metabolismo , Methylococcus capsulatus/metabolismo , Methylosinus trichosporium/metabolismo , Estructura MolecularRESUMEN
Methane-oxidizing bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase. Certain methanotrophs are also able to switch to using the iron-containing soluble methane monooxygenase to catalyse methane oxidation, with this switchover regulated by copper. Methane monooxygenases are nature's primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and methane monooxygenases have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. Here we discover and characterize a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for particulate methane monooxygenase. Csp1 is a tetramer of four-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realized. Cytosolic homologues of Csp1 are present in diverse bacteria, thus challenging the dogma that such organisms do not use copper in this location.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Metano/metabolismo , Methylosinus trichosporium/química , Secuencias de Aminoácidos , Cristalografía por Rayos X , Citosol/metabolismo , Metano/química , Methylosinus trichosporium/enzimología , Modelos Moleculares , Oxidación-Reducción , Oxigenasas/metabolismo , Pliegue de Proteína , Estructura Secundaria de ProteínaRESUMEN
Soluble methane monooxygenase (sMMO) is a bacterial multicomponent enzyme that oxidizes a diverse range of substrates, including aromatic hydrocarbons. We have investigated enzyme-substrate interactions that govern oxidation regioselectivity at various sites of aromatic compounds using substrate docking and molecular dynamics (MD) simulations. Here, we studied the hydroxylation of toluene and ethyl benzene by two forms of Methylosinus trichosporium OB3b (sMMO), that is, wild-type (WT) and two active site mutants (L110Y/G). The two substrates, toluene and ethyl benzene, were docked into the active site of the WT and the L110Y/G mutant models of M. trichosporium OB3b sMMO using the available X-ray structure (PDB id 1 MHZ). The trends observed in the formation of the experimental product were highly correlated with the results obtained from the relatively short MD simulation. These results show that our approach could be an attractive computational tool to rationalize the prediction of product ratios and specificities.
Asunto(s)
Hidrocarburos Aromáticos/metabolismo , Simulación de Dinámica Molecular , Oxigenasas/metabolismo , Hidrocarburos Aromáticos/química , Hidroxilación , Methylosinus trichosporium/química , Modelos Moleculares , Estructura Molecular , Oxigenasas/química , Solubilidad , EstereoisomerismoRESUMEN
Methanobactin (mb) is a post-translationally modified copper-binding compound, or chalkophore, secreted by many methane-oxidizing bacteria or methanotrophs in response to copper limitation. In addition to copper, methanobactin from Methylosinus trichosporium OB3b (mb-OB3b) has been shown to bind a variety of metals including Hg(2+). In this report, Hg binding by the structurally unique methanobactin from Methylocystis strain SB2 (mb-SB2) was examined and compared to mb-OB3b. Mb-SB2 is shown to bind the common forms of Hg found in aqueous environments, Hg(2+), Hg(CN)2 and CH3Hg(+). The spectral and thermodynamic properties of binding for each form of mercury differed. UV-visible absorption spectra suggested that Hg(2+) binds to both the oxazolone and imidazolone rings of mb-SB2, whereas CH3Hg(+) appeared to only bind to the oxazolone ring. Hg(CN)2 showed spectral properties between Hg(2+) and CH3Hg(+). Isothermal titration calorimetry (ITC) showed both Hg(CN)2 and CH3Hg(+) fit into two-site binding models. For Hg(CN)2 the first site was exothermic and the second endothermic. Both binding sites in CH3Hg(+) were exothermic, but at equilibrium the reaction never moved back to the baseline, suggesting a slow residual reaction. ITC results for Hg(2+) were more complex and suggested a 3- or 4-site model. The spectral, kinetic and thermodynamic changes following Hg binding by mb-SB2 also differed from the changes associated with mb-OB3b. Like mb-OB3b, copper did not displace Hg bound to mb-SB2. In contrast to mb-OB3b Hg(2+) could displace Cu from Cu-containing mb-SB2 and preferentially bound Hg(2+) over Cu(2+) at metal to mb-SB2 molar ratios above 1.0.
Asunto(s)
Proteínas Bacterianas/química , Cobre/química , Imidazoles/química , Mercurio/química , Methylocystaceae/química , Oligopéptidos/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cationes Bivalentes , Cobre/metabolismo , Expresión Génica , Imidazoles/aislamiento & purificación , Imidazoles/metabolismo , Cinética , Mercurio/metabolismo , Methylocystaceae/crecimiento & desarrollo , Methylocystaceae/metabolismo , Methylosinus trichosporium/química , Oligopéptidos/aislamiento & purificación , Oligopéptidos/metabolismo , Unión Proteica , TermodinámicaRESUMEN
Copper has been found to play a key role in the physiology of methanotrophic micro-organisms, and methane oxidation may critically depend on the availability of Cu. In natural environments, such as soils, sediments, peat bogs, and surface waters, the presence of natural organic matter (NOM) can control the bioavailability of Cu by forming strong metal complexes. To promote Cu acquisition, methanotrophs exude methanobactin, a ligand known to have a high affinity for Cu. In this study, the capability of methanobactin for Cu acquisition from NOM was investigated using humic acid (HA) as a model substance. The kinetics of ligand exchange between Cu-HA and methanobactin was observed by UV-vis spectroscopy, and the speciation of Cu bound to methanobactin was determined by size-exclusion chromatography coupled to an ICP-MS. The results showed that Cu was mobilized from HA by a fast ligand exchange reaction following a second-order rate law with first-order kinetics for both methanobactin and Cu-HA complexes. The reaction rates decreased with decreasing temperature. Equilibrium experiments indicated that methanobactin was not sorbed to HA and proved that methanobactin is competitive with HA for Cu binding by forming strong 1:1 Cu-methanobactin complexes. Consequently, our results demonstrate that methanobactin can efficiently acquire Cu in organic-rich environments.
Asunto(s)
Cobre/metabolismo , Sustancias Húmicas , Imidazoles/metabolismo , Methylosinus trichosporium/metabolismo , Oligopéptidos/metabolismo , Cromatografía en Gel , Cobre/química , Imidazoles/química , Ligandos , Espectrometría de Masas , Methylosinus trichosporium/química , Oligopéptidos/química , TemperaturaRESUMEN
Methanobactins are copper-binding ligands produced by aerobic methanotrophic microorganisms. A quantitative understanding of their potential role in methanotrophic copper acquisition requires the investigation of their copper complexes under relevant pH conditions. In this study, a chemical speciation model describing the pH-dependence of copper binding and the formation of the different complexes by methanobactin (mb) is released by Methylosinus trichosporium OB3b was developed. Potentiometric and spectrophotometric titrations of the free ligand indicated the presence of four protonation sites consistent with the molecular structure of methanobactin. Metal titrations revealed a distinct pH-dependence of copper binding to methanobactin between pH 5 and 8. Based on evidence from size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS), the copper binding was quantitatively described with three different types of copper-methanobactin complexes which can additionally undergo protonation reactions. The high affinity observed upon initial copper additions resulted from the predominant occurrence of copper-methanobactin dimer complexes, mb(2)H(4)Cu and mb(2)H(3)Cu with log K values of 58 and 52, respectively. With increasing copper to methanobactin ratios, methanobactin bound copper as monomers, mbHCu (log K=25) and mbCu (log K=18), whereas at elevated copper activities methanobactin was able to bind two copper ions (mbHCu(2) and mbCu(2)). Model calculations based on the fitted complexation constants suggest that in natural systems, copper-methanobactin complexes are mostly present as monomers.
Asunto(s)
Cobre/química , Concentración de Iones de Hidrógeno , Imidazoles/química , Methylosinus trichosporium/química , Modelos Teóricos , Oligopéptidos/química , Cromatografía en Gel , PotenciometríaRESUMEN
The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His(6)-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.
Asunto(s)
Proteínas Bacterianas/química , Methylobacterium/enzimología , Methylosinus trichosporium/enzimología , Fosfofructoquinasa-1/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Fructosafosfatos/metabolismo , Cinética , Methylobacterium/química , Methylobacterium/clasificación , Methylobacterium/genética , Methylosinus trichosporium/química , Methylosinus trichosporium/clasificación , Methylosinus trichosporium/genética , Datos de Secuencia Molecular , Fosfofructoquinasa-1/genética , Fosfofructoquinasa-1/metabolismo , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Methanobactins (mbs) are Low molecular mass copper binding chromopeptides analogous to pyoverdin class iron-binding siderophores. Mb produced by Methylosinus trichosporium OB3b (mb-oB3b) has been used as a model molecuLe for methanobactin although the amino acid sequence of mb-OB3b differs significantly from other characterized mbs. In particular, there is the presence of a pair of cystine residues which are absent in other characterized mbs. The role of the Cys3-Cys6 in copper binding, Cu(ll) reduction and its role on the mb-OB3b structure remains in debate. Here, we use a single-step dithiothreitol treatment as an effective method in reducing the disulfide bond allowing in-depth ion mobility-mass spectrometry (IM-MS) analysis. The IM-MS results show mb-oB3b exists in the gas-phase as three different negatively-charged states and exists in multiple conformational states, when introduced via electrospray ionization from aqueous solution near physiological pH. The disulfide bond serves a structural role and is not involved in the Cu(I/ll) binding capability of mb-OB3b, with the binding of a second Cu(I/ll) related to a further deprotonation of mb-OB3b. Overall, these findings are in good correlation with expected solution-phase behavior of mb-OB3b. The results suggest IM-MS is an effective tool for better understanding the complex nature of this intriguing peptide.
Asunto(s)
Cobre/metabolismo , Imidazoles/química , Methylosinus trichosporium/química , Oligopéptidos/química , Espectrometría de Masa por Ionización de Electrospray , Sitios de Unión , Disulfuros/química , Ditiotreitol/química , Diseño de Equipo , Imidazoles/aislamiento & purificación , Imidazoles/metabolismo , Methylosinus trichosporium/metabolismo , Oligopéptidos/aislamiento & purificación , Oligopéptidos/metabolismo , Oxidación-Reducción , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Methanotrophic bacteria, organisms that oxidize methane, produce a small copper chelating molecule called methanobactin (Mb). Mb binds Cu(I) with high affinity and is hypothesized to mediate copper acquisition from the environment. Recent advances in Mb characterization include revision of the chemical structure of Mb from Methylosinus trichosporium OB3b and further investigation of its biophysical properties. In addition, Mb production by several other methanotroph strains has been investigated, and preliminary characterization suggests diversity in chemical composition. Initial clues into Mb biosynthesis have been obtained by identification of a putative precursor gene in the M. trichosporium OB3b genome. Finally, direct uptake of intact Mb into the cytoplasm of M. trichosporium OB3b cells has been demonstrated, and studies of the transport mechanism have been initiated. Taken together, these advances represent significant progress and set the stage for exciting new research directions.
Asunto(s)
Quelantes/metabolismo , Cobre/metabolismo , Imidazoles/metabolismo , Methylosinus trichosporium/metabolismo , Oligopéptidos/metabolismo , Quelantes/química , Cobre/química , Genes Bacterianos , Imidazoles/química , Methylosinus trichosporium/química , Methylosinus trichosporium/genética , Oligopéptidos/química , Oligopéptidos/genéticaRESUMEN
Particulate methane monooxygenase (pMMO) catalyzes methane hydroxylation to methanol at ambient temperature and pressure. pMMO from Methylosinus trichosporium OB3b is one of the two pMMOs for which the protein structure was determined by X-ray crystallography. Because purified pMMO is inherently instable in vitro, it is difficult to use for time-consuming analysis. Therefore, investigations using crude enzyme preparations of pMMO are useful in some cases. In this chapter, methods for preparing pMMO from M. trichosporium OB3b to varying degrees of purity, including bacterial cells expressing pMMO, membrane fractions containing pMMO, and highly purified pMMO, are described.
Asunto(s)
Methylosinus trichosporium/enzimología , Oxigenasas/aislamiento & purificación , Oxigenasas/metabolismo , Alcanos/metabolismo , Alquenos/metabolismo , Cromatografía/métodos , Cristalografía por Rayos X , Detergentes , Metales/análisis , Metanol/metabolismo , Methylosinus trichosporium/química , Methylosinus trichosporium/metabolismo , Oxigenasas/química , SolubilidadRESUMEN
Aerobic methane-oxidizing bacteria (methanotrophs) have a high conditional need for copper because almost all known species express a copper-containing particulate methane monooxygenase for catalyzing the conversion of methane to methanol. This demands a copper homeostatic system that must both supply and satisfy adequate copper for elevated needs while also shielding the cells from copper toxicity. After considerable effort, it was discovered that some methanotrophs produce small peptidic molecules, called methanobactins, which bind copper, mediate copper transport into the cell, and reduce copper toxicity. Unfortunately, isolating, purifying, and proving the functionality of these molecules has been challenging. In fact, until very recently, only one complete structure had been reported for methanobactins. As such, there is a desperate need for more studies seeking such molecules. The purpose of this chapter is to describe methods used to isolate and purify the original methanobactin with a published complete structure, which is made by Methylosinus trichosporium OB3b. Methods are also included for assessing the function of such molecules under pseudonatural conditions such as growth on mineral copper sources. Special emphasis is placed on verifying that isolated molecules are "true" methanobactins, because recent work has shown that methanotrophs produce other small molecules that also bind metals in solution.
Asunto(s)
Cobre/metabolismo , Imidazoles/aislamiento & purificación , Imidazoles/metabolismo , Methylosinus trichosporium/metabolismo , Oligopéptidos/aislamiento & purificación , Oligopéptidos/metabolismo , Imidazoles/química , Espectrometría de Masas/métodos , Methylosinus trichosporium/química , Methylosinus trichosporium/crecimiento & desarrollo , Oligopéptidos/química , Unión ProteicaRESUMEN
Chalkophores are low molecular mass modified peptides involved in copper acquisition in methane-oxidizing bacteria (MOB). A screening method for the detection of this copper-binding molecule is presented in Chapter 16. Here we describe methods to (1) maximize expression and secretion of chalkophores, (2) concentrate chalkophores from the spent media of MOB, and (3) purify chalkophores.
Asunto(s)
Imidazoles/aislamiento & purificación , Methylobacterium/química , Methylococcus capsulatus/química , Methylocystaceae/química , Methylosinus trichosporium/química , Oligopéptidos/aislamiento & purificaciónRESUMEN
Methanobactin (mb) is a low molecular mass copper-binding molecule analogous to iron-binding siderophores. The molecule is produced by many methanotrophic or methane oxidizing bacteria (MOB), but has only been characterized to date in one MOB, Methylosinus trichosporium OB3b. To explore the potential molecular diversity in this novel class of metal binding compound, the spectral (UV-visible, fluorescent, and electron paramagnetic resonance) and thermodynamic properties of mb from two γ-proteobacterial MOB, Methylococcus capsulatus Bath and Methylomicrobium album BG8, were determined and compared to the mb from the α-proteobacterial MOB, M. trichosporium OB3b. The mb from both γ-proteobacterial MOB differed from the mb from M. trichosporium OB3b in molecular mass and spectral properties. Compared to mb from M. trichosporium OB3b, the extracellular concentrations were low, as were copper-binding constants of mb from both γ-proteobacterial MOB. In addition, the mb from M. trichosporium OB3b removed Cu(I) from the mb of both γ-proteobacterial MOB. Taken together the results suggest mb may be a factor in regulating methanotrophic community structure in copper-limited environments.