RESUMO
Methanobactins (MBs) are small (<1,300-Da) posttranslationally modified copper-binding peptides and represent the extracellular component of a copper acquisition system in some methanotrophs. Interestingly, MBs can bind a range of metal ions, with some being reduced after binding, e.g., Cu2+ reduced to Cu+. Other metal ions, however, are bound but not reduced, e.g., K+. The source of electrons for selective metal ion reduction has been speculated to be water but never empirically shown. Here, using H218O, we show that when MBs from Methylocystis sp. strain SB2 (MB-SB2) and Methylosinus trichosporium OB3b (MB-OB3) were incubated in the presence of either Au3+, Cu2, or Ag+, 18,18O2 and free protons were released. No 18,18O2 production was observed in the presence of either MB-SB2 or MB-OB3b alone, gold alone, copper alone, or silver alone or when K+ or Mo2+ was incubated with MB-SB2. In contrast to MB-OB3b, MB-SB2 binds Fe3+ with an N2S2 coordination and will also reduce Fe3+ to Fe2+. Iron reduction was also found to be coupled to the oxidation of 2H2O and the generation of O2. MB-SB2 will also couple Hg2+, Ni2+, and Co2+ reduction to the oxidation of 2H2O and the generation of O2, but MB-OB3b will not, ostensibly as MB-OB3b binds but does not reduce these metal ions. To determine if the O2 generated during metal ion reduction by MB could be coupled to methane oxidation, 13CH4 oxidation by Methylosinus trichosporium OB3b was monitored under anoxic conditions. The results demonstrate that O2 generation from metal ion reduction by MB-OB3b can support methane oxidation. IMPORTANCE The discovery that MB will couple the oxidation of H2O to metal ion reduction and the release of O2 suggests that methanotrophs expressing MB may be able to maintain their activity under hypoxic/anoxic conditions through the "self-generation" of dioxygen required for the initial oxidation of methane to methanol. Such an ability may be an important factor in enabling methanotrophs to not only colonize the oxic-anoxic interface where methane concentrations are highest but also tolerate significant temporal fluctuations of this interface. Given that genomic surveys often show evidence of aerobic methanotrophs within anoxic zones, the ability to express MB (and thereby generate dioxygen) may be an important parameter in facilitating their ability to remove methane, a potent greenhouse gas, before it enters the atmosphere.
Assuntos
Imidazóis/metabolismo , Metais Pesados/metabolismo , Metano/metabolismo , Methylocystaceae/metabolismo , Oligopeptídeos/metabolismo , Oxigênio/química , Água/química , Metais Pesados/química , OxirreduçãoRESUMO
In Wilson disease (WD), functional loss of ATPase copper-transporting ß (ATP7B) impairs biliary copper excretion, leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration- and European Medicines Agency-approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure, leaving liver transplantation as the only viable treatment option. Here, we investigated the therapeutic utility of methanobactin (MB), a peptide produced by Methylosinus trichosporium OB3b, which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes, including hepatic copper accumulation, liver damage, and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover, MB treatment prevented hepatocyte death, subsequent liver failure, and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD.
Assuntos
Degeneração Hepatolenticular/tratamento farmacológico , Imidazóis/farmacologia , Falência Hepática Aguda/tratamento farmacológico , Oligopeptídeos/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Bile/química , Proteínas de Transporte de Cátions/metabolismo , Quelantes/química , Cobre/química , ATPases Transportadoras de Cobre , Modelos Animais de Doenças , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fenótipo , RatosRESUMO
Methanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin, mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well as mbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT is truly responsible for methanobactin uptake, a knockout was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT::Gm(r) mutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-type M. trichosporium OB3b, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT::Gm(r) mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-type M. trichosporium OB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.
Assuntos
Proteínas de Bactérias/metabolismo , Imidazóis/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Methylosinus trichosporium/metabolismo , Oligopeptídeos/metabolismo , Cobre/metabolismo , Técnicas de Inativação de Genes , Proteínas de Membrana Transportadoras/genética , Methylosinus trichosporium/genéticaRESUMO
Methanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a "copper switch." At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO.
Assuntos
Oxirredutases do Álcool/biossíntese , Cério/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Expressão Gênica , Methylosinus trichosporium/efeitos dos fármacos , Methylosinus trichosporium/enzimologia , Cobre/metabolismo , Meios de Cultura/química , Metano/metabolismo , Methylosinus trichosporium/genética , Methylosinus trichosporium/crescimento & desenvolvimento , OxirreduçãoRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Cobre/química , Imidazóis/química , Mercúrio/química , Methylocystaceae/química , Oligopeptídeos/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cátions Bivalentes , Cobre/metabolismo , Expressão Gênica , Imidazóis/isolamento & purificação , Imidazóis/metabolismo , Cinética , Mercúrio/metabolismo , Methylocystaceae/crescimento & desenvolvimento , Methylocystaceae/metabolismo , Methylosinus trichosporium/química , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/metabolismo , Ligação Proteica , TermodinâmicaRESUMO
Many methanotrophs have been shown to synthesize methanobactin, a novel biogenic copper-chelating agent or chalkophore. Methanobactin binds copper via two heterocyclic rings with associated enethiol groups. The structure of methanobactin suggests that it can bind other metals, including mercury. Here we report that methanobactin from Methylosinus trichosporium OB3b does indeed bind mercury when added as HgCl2 and, in doing so, reduced toxicity associated with Hg(II) for both Alphaproteobacteria methanotrophs, including M. trichosporium OB3b, M. trichosporium OB3b ΔmbnA (a mutant defective in methanobactin production), and Methylocystis sp. strain SB2, and a Gammaproteobacteria methanotroph, Methylomicrobium album BG8. Mercury binding by methanobactin was evident in both the presence and absence of copper, despite the fact that methanobactin had a much higher affinity for copper due to the rapid and irreversible binding of mercury by methanobactin. The formation of a gray precipitate suggested that Hg(II), after being bound by methanobactin, was reduced to Hg(0) but was not volatilized. Rather, mercury remained associated with methanobactin and was also found associated with methanotrophic biomass. It thus appears that although the mercury-methanobactin complex was cell associated, mercury was not removed from methanobactin. The amount of biomass-associated mercury in the presence of methanobactin from M. trichosporium OB3b was greatest for M. trichosporium wild-type strain OB3b and the ΔmbnA mutant and least for M. album BG8, suggesting that methanotrophs may have selective methanobactin uptake systems that may be based on TonB-dependent transporters but that such uptake systems exhibit a degree of infidelity.
Assuntos
Imidazóis/metabolismo , Cloreto de Mercúrio/metabolismo , Cloreto de Mercúrio/toxicidade , Methylosinus trichosporium/efeitos dos fármacos , Methylosinus trichosporium/metabolismo , Oligopeptídeos/metabolismo , Alphaproteobacteria/efeitos dos fármacos , Inativação Metabólica , Methylococcaceae/efeitos dos fármacos , Methylocystaceae/efeitos dos fármacos , OxirreduçãoRESUMO
Biological oxidation of methane to methanol by aerobic bacteria is catalysed by two different enzymes, the cytoplasmic or soluble methane monooxygenase (sMMO) and the membrane-bound or particulate methane monooxygenase (pMMO). Expression of MMOs is controlled by a 'copper-switch', i.e. sMMO is only expressed at very low copper : biomass ratios, while pMMO expression increases as this ratio increases. Methanotrophs synthesize a chalkophore, methanobactin, for the binding and import of copper. Previous work suggested that methanobactin was formed from a polypeptide precursor. Here we report that deletion of the gene suspected to encode for this precursor, mbnA, in Methylosinus trichosporium OB3b, abolishes methanobactin production. Further, gene expression assays indicate that methanobactin, together with another polypeptide of previously unknown function, MmoD, play key roles in regulating expression of MMOs. Based on these data, we propose a general model explaining how expression of the MMO operons is regulated by copper, methanobactin and MmoD. The basis of the 'copper-switch' is MmoD, and methanobactin amplifies the magnitude of the switch. Bioinformatic analysis of bacterial genomes indicates that the production of methanobactin-like compounds is not confined to methanotrophs, suggesting that its use as a metal-binding agent and/or role in gene regulation may be widespread in nature.
Assuntos
Cobre/metabolismo , Imidazóis/metabolismo , Methylosinus trichosporium/genética , Oligopeptídeos/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Transporte Biológico , Deleção de Genes , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Metano/metabolismo , Metanol/metabolismo , Methylosinus trichosporium/metabolismo , Oligopeptídeos/biossíntese , Óperon , Oxirredução , Oxigenases/biossínteseRESUMO
Methanobactin (mb) is the first characterized example of a chalkophore, a class of copper-binding chromopeptides similar to iron-binding siderophores. Structural, redox, themodynamic, and spectral studies on chalkophores have focused almost exclusively on the mb from Methylosinus trichosporium OB3b (mb-OB3b). The structural characterization of a second mb from Methylocystis strain SB2 (mb-SB2) provides a means to examine the core structural features and metal binding properties of this group of chromopeptides. With the exception of the 5-membered rings (either oxazolone or imidazolone), enethiol groups, and the N-terminus oxo group, the structure of mb-SB2 differs markedly from mb-OB3b. In particular the amino acids commonly associated with metal coordination and redox activity found in mb-OB3b, Cys, Met, and Try, are replaced by Ala or are missing in mb-SB2. In this report the spectral and thermodynamic properties of mb-SB2 are presented and compared to mb-OB3b. The results demonstrate that the spectral and basic copper binding properties of both methanobactins are similar and the unique copper binding capacity of both methanobactins lies primarily in the pair of five-membered rings and associated enethiol groups. The remaining portions of the methanobactin appear to provide the scaffolding that brings together of the two ring systems to produce the tetrahedral binding site for copper binding.
Assuntos
Proteínas de Bactérias/química , Cobre/química , Imidazóis/química , Methylocystaceae/química , Oligopeptídeos/química , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Imidazóis/metabolismo , Methylocystaceae/metabolismo , Oligopeptídeos/metabolismo , Oxirredução , Ligação Proteica , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Imidazóis/isolamento & purificação , Methylobacterium/química , Methylococcus capsulatus/química , Methylocystaceae/química , Methylosinus trichosporium/química , Oligopeptídeos/isolamento & purificaçãoRESUMO
Wilson disease (WD) is a rare hereditary condition that is caused by a genetic defect in the copper-transporting ATPase ATP7B that results in hepatic copper accumulation and lethal liver failure. The present study focuses on the structural mitochondrial alterations that precede clinical symptoms in the livers of rats lacking Atp7b, an animal model for WD. Liver mitochondria from these Atp7b/ rats contained enlarged cristae and widened intermembrane spaces, which coincided with a massive mitochondrial accumulation of copper. These changes, however, preceded detectable deficits in oxidative phosphorylation and biochemical signs of oxidative damage, suggesting that the ultrastructural modifications were not the result of oxidative stress imposed by copper- dependent Fenton chemistry. In a cell-free system containing a reducing dithiol agent, isolated mitochondria exposed to copper underwent modifications that were closely related to those observed in vivo. In this cell-free system, copper induced thiol modifications of three abundant mitochondrial membrane proteins, and this correlated with reversible intramitochondrial membrane crosslinking, which was also observed in liver mitochondria from Atp7b/ rats. In vivo, copper-chelating agents reversed mitochondrial accumulation of copper, as well as signs of intra-mitochondrial membrane crosslinking, thereby preserving the functional and structural integrity of mitochondria. Together, these findings suggest that the mitochondrion constitutes a pivotal target of copper in WD.
Assuntos
Degeneração Hepatolenticular/patologia , Mitocôndrias Hepáticas/patologia , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Proteínas de Transporte de Cátions , Sistema Livre de Células , Quelantes/farmacologia , Cobre/metabolismo , ATPases Transportadoras de Cobre , Reagentes de Ligações Cruzadas , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Degeneração Hepatolenticular/genética , Degeneração Hepatolenticular/metabolismo , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Transmissão , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Ratos , Compostos de Sulfidrila/metabolismoRESUMO
Copper is an essential redox-active metal ion which in excess becomes toxic due to the formation of reactive oxygen species. In Wilson disease the elevated copper level in liver leads to chronic oxidative stress and subsequent hepatitis. This study was designed to evaluate the copper chelating efficiency of the bacterial methanobactin (MB) in a rat model for Wilson disease. Methanobactin is a small peptide produced by the methanotrophic bacterium Methylosinus trichosporium OB3b and has an extremely high affinity for copper. Methanobactin treatment of the rats was started at high liver copper and serum aspartate aminotransferase (AST) levels. Two dosing schedules with either 6 or 13 intraperitoneal doses of 200mg methanobactin per kg body weight were applied. Methanobactin treatment led to a return of serum AST values to basal levels and a normalization of liver histopathology. Concomitantly, copper levels declined to 45% and 24% of untreated animals after 6 and 13 doses, respectively. Intravenous application of methanobactin led to a prompt release of copper from liver into bile and the copper was shown to be associated with methanobactin. In vitro experiments with liver cytosol high in copper metallothionein demonstrated that methanobactin removes copper from metallothionein confirming the potent copper chelating activity of methanobactin.
Assuntos
Quelantes/uso terapêutico , Modelos Animais de Doenças , Degeneração Hepatolenticular/tratamento farmacológico , Imidazóis/uso terapêutico , Oligopeptídeos/uso terapêutico , Animais , Cromatografia Líquida de Alta Pressão , Ratos , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria UltravioletaRESUMO
The particulate methane monooxygenase (pMMO) in Methylocystis strain SB2 was found to be constitutively expressed in the absence of methane when the strain was grown on either acetate or ethanol. Real-time quantitative polymerase chain reaction (PCR) and reverse transcription-PCR showed that the expression of pmoA decreased by one to two orders of magnitude when grown on acetate as compared with growth of strain SB2 on methane. The capability of strain SB2 to degrade a mixture of chlorinated ethenes in the absence of methane was examined to verify the presence and activity of pMMO under acetate-growth conditions as well determine the effectiveness of such conditions for bioremediation. It was found that when strain SB2 was grown on acetate and exposed to 40 µM each of trichloroethylene (TCE), trans-dichloroethylene (t-DCE) and vinyl chloride (VC), approximately 30% of VC and t-DCE was degraded but no appreciable TCE removal was measured after 216 h of incubation. The ability to degrade VC and t-DCE was lost when acetylene was added, confirming that pMMO was responsible for the degradation of these chlorinated ethenes by Methylocystis strain SB2 when the strain was grown on acetate.
RESUMO
Methanobactins (mb) are low-molecular mass, copper-binding molecules secreted by most methanotrophic bacteria. These molecules have been identified for a number of methanotrophs, but only the one produced by Methylosinus trichosporium OB3b (mb-OB3b) has to date been chemically characterized. Here we report the chemical characterization and copper binding properties of a second methanobactin, which is produced by Methylocystis strain SB2 (mb-SB2). mb-SB2 shows some significant similarities to mb-OB3b, including its spectral and metal binding properties, and its ability to bind and reduce Cu(II) to Cu(I). Like mb-OB3b, mb-SB2 contains two five-member heterocyclic rings with associated enethiol groups, which together form the copper ion binding site. mb-SB2 also displays some significant differences compared to mb-OB3b, including the number and types of amino acids used to complete the structure of the molecule, the presence of an imidazolone ring in place of one of the oxazolone rings found in mb-OB3b, and the presence of a sulfate group not found in mb-OB3b. The sulfate is bonded to a threonine-like side chain that is associated with one of the heterocyclic rings and may represent the first example of this type of sulfate group found in a bacterially derived peptide. Acid-catalyzed hydrolysis and decarboxylation of the oxazolone rings found in mb-OB3b and mb-SB2 produce pairs of amino acid residues and suggest that both mb-OB3b and mb-SB2 are derived from peptides. In support of this, the gene for a ribosomally produced peptide precursor for mb-OB3b has been identified in the genome of M. trichosporium OB3b. The gene sequence indicates that the oxazolone rings in mb-OB3b are derived from the combination of a cysteine residue and the carbonyl from the preceding residue in the peptide sequence. Taken together, the results suggest methanobactins make up a structurally diverse group of ribosomally produced, peptide-derived molecules, which share a common pair of five-member rings with associated enethiol groups that are able to bind, reduce, and stabilize copper ions in an aqueous environment.
Assuntos
Cobre/metabolismo , Imidazóis/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Methylocystaceae/metabolismo , Methylosinus trichosporium/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/biossíntese , Espectrofotometria UltravioletaRESUMO
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.