RESUMO
Methanobactins (Mbns) are ribosomally synthesized and posttranslationally modified peptide natural products released by methanotrophic bacteria under conditions of copper scarcity. Mbns bind Cu(I) with high affinity via nitrogen-containing heterocycles and thioamide groups installed on a precursor peptide, MbnA, by a core biosynthetic enzyme complex, MbnBC. Additional stabilizing modifications are enacted by other, less universal biosynthetic enzymes. Copper-loaded Mbn is imported into the cell by TonB-dependent transporters called MbnTs, and copper is mobilized by an unknown mechanism. The machinery to biosynthesize and transport Mbn is encoded in operons that are also found in the genomes of nonmethanotrophic bacteria. In this review, we provide an update on the state of the Mbn field, highlighting recent discoveries regarding Mbn structure, biosynthesis, and handling as well as the emerging roles of Mbns in the environment and their potential use as therapeutics.
RESUMO
The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.
Assuntos
Proteínas de Bactérias , Cobre , Haemophilus influenzae , Oxazolona , Fatores de Virulência , Haemophilus influenzae/metabolismo , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Haemophilus influenzae/patogenicidade , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Cobre/metabolismo , Cobre/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Oxazolona/metabolismo , Tioamidas/metabolismo , Tioamidas/química , Ferro/metabolismo , Processamento de Proteína Pós-Traducional , Oxirredutases/metabolismo , Oxirredutases/genética , Óperon , Cisteína/metabolismoRESUMO
The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.
Assuntos
Imidazóis , Oligopeptídeos , Imidazóis/metabolismo , Imidazóis/química , Oligopeptídeos/metabolismo , Oligopeptídeos/química , Oligopeptídeos/biossíntese , Oxirredução , Cristalografia por Raios X , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Espectroscopia de Ressonância de Spin Eletrônica , Oxigenases/metabolismo , Oxigenases/química , Domínio Catalítico , Especificidade por Substrato , Modelos Moleculares , Ferro/metabolismo , Ferro/químicaRESUMO
Methane is a potent greenhouse gas that contributes significantly to climate change and is primarily regulated in Nature by methanotrophic bacteria, which consume methane gas as their source of energy and carbon, first by oxidizing it to methanol. The direct oxidation of methane to methanol is a chemically difficult transformation, accomplished in methanotrophs by complex methane monooxygenase (MMO) enzyme systems. These enzymes use iron or copper metallocofactors and have been the subject of detailed investigation. While the structure, function, and active site architecture of the copper-dependent particulate methane monooxygenase (pMMO) have been investigated extensively, its putative quaternary interactions, regulation, requisite cofactors, and mechanism remain enigmatic. The iron-dependent soluble methane monooxygenase (sMMO) has been characterized biochemically, structurally, spectroscopically, and, for the most part, mechanistically. Here, we review the history of MMO research, focusing on recent developments and providing an outlook for future directions of the field. Engineered biological catalysis systems and bioinspired synthetic catalysts may continue to emerge along with a deeper understanding of the molecular mechanisms of biological methane oxidation. Harnessing the power of these enzymes will necessitate combined efforts in biochemistry, structural biology, inorganic chemistry, microbiology, computational biology, and engineering.
Assuntos
Cobre , Metano , Cobre/química , Ferro , Metanol , Oxigenases/metabolismo , Oxirredução , Oxigenases de Função MistaRESUMO
The diheme bacterial cytochrome c peroxidase (bCcP)/MauG superfamily is a diverse set of enzymes that remains largely uncharacterized. One recently discovered member, MbnH, converts a tryptophan residue in its substrate protein, MbnP, to kynurenine. Here we show that upon reaction with H2O2, MbnH forms a bis-Fe(IV) intermediate, a state previously detected in just two other enzymes, MauG and BthA. Using absorption, Mössbauer, and electron paramagnetic resonance (EPR) spectroscopies coupled with kinetic analysis, we characterized the bis-Fe(IV) state of MbnH and determined that this intermediate decays back to the diferric state in the absence of MbnP substrate. In the absence of MbnP substrate, MbnH can also detoxify H2O2 to prevent oxidative self damage, unlike MauG, which has long been viewed as the prototype for bis-Fe(IV) forming enzymes. MbnH performs a different reaction from MauG, while the role of BthA remains unclear. All three enzymes can form a bis-Fe(IV) intermediate but within distinct kinetic regimes. The study of MbnH significantly expands our knowledge of enzymes that form this species. Computational and structural analyses indicate that electron transfer between the two heme groups in MbnH and between MbnH and the target tryptophan in MbnP likely occurs via a hole-hopping mechanism involving intervening tryptophan residues. These findings set the stage for discovery of additional functional and mechanistic diversity within the bCcP/MauG superfamily.
Assuntos
Methylosinus trichosporium , Methylosinus trichosporium/metabolismo , Triptofano/química , Cinética , Peróxido de Hidrogênio/metabolismo , Oxirredução , Bactérias/metabolismoRESUMO
Nature's primary methane-oxidizing enzyme, the membrane-bound particulate methane monooxygenase (pMMO), catalyzes the oxidation of methane to methanol. pMMO activity requires copper, and decades of structural and spectroscopic studies have sought to identify the active site among three candidates: the CuB, CuC, and CuD sites. Challenges associated with the isolation of active pMMO have hindered progress toward locating its catalytic center. However, reconstituting pMMO into native lipid nanodiscs stabilizes its structure and recovers its activity. Here, these active samples were incubated with 2,2,2,-trifluoroethanol (TFE), a product analog that serves as a readily visualized active-site probe. Interactions of TFE with the CuD site were observed by both pulsed ENDOR spectroscopy and cryoEM, implicating CuD and the surrounding hydrophobic pocket as the likely site of methane oxidation. Use of these orthogonal techniques on parallel samples is a powerful approach that can circumvent difficulties in interpreting metalloenzyme cryoEM maps.
RESUMO
Particulate methane monooxygenase (pMMO) is a multi-subunit membrane metalloenzyme used by methanotrophic bacteria to convert methane to methanol. A major hurdle to studying pMMO is the lack of a recombinant expression system, precluding investigation of individual residues by mutagenesis and hampering a complete understanding of its mechanism. Here, we developed an Escherichia coli lysate-based cell-free protein synthesis (CFPS) system that can be used to express pMMO in vitro in the presence of nanodiscs. We used a SUMO fusion construct to generate the native PmoB subunit and showed that the SUMO protease (Ulp1) cleaves the protein in the reaction mixture. Using an affinity tag to isolate the complete pMMO complex, we demonstrated that the complex forms without the need for exogenous translocon machinery or chaperones, confirmed by negative stain electron microscopy. This work demonstrates the potential for using CFPS to express multi-subunit membrane-bound metalloenzymes directly into lipid bilayers.
Assuntos
Methylococcus capsulatus , Methylococcus capsulatus/metabolismo , Oxirredução , Cobre/química , Cobre/metabolismo , Oxigenases/metabolismoRESUMO
Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.
Assuntos
Methylococcaceae , Oxigenases , Cobre/química , Metano/metabolismo , Methylococcaceae/metabolismo , Minerais , Oxirredução , Oxigenases/metabolismoRESUMO
Triosephosphate isomerase (TPI) performs the 5th step in glycolysis, operates near the limit of diffusion, and is involved in "moonlighting" functions. Its dimer was found singly phosphorylated at Ser20 (pSer20) in human cells, with this post-translational modification (PTM) showing context-dependent stoichiometry and loss under oxidative stress. We generated synthetic pSer20 proteoforms using cell-free protein synthesis that showed enhanced TPI activity by 4-fold relative to unmodified TPI. Molecular dynamics simulations show that the phosphorylation enables a channel to form that shuttles substrate into the active site. Refolding, kinetic, and crystallographic analyses of point mutants including S20E/G/Q indicate that hetero-dimerization and subunit asymmetry are key features of TPI. Moreover, characterization of an endogenous human TPI tetramer also implicates tetramerization in enzymatic regulation. S20 is highly conserved across eukaryotic TPI, yet most prokaryotes contain E/D at this site, suggesting that phosphorylation of human TPI evolved a new switch to optionally boost an already fast enzyme. Overall, complete characterization of TPI shows how endogenous proteoform discovery can prioritize functional versus bystander PTMs.
Assuntos
Simulação de Dinâmica Molecular , Triose-Fosfato Isomerase , Humanos , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismo , Fosforilação , Domínio Catalítico , CinéticaRESUMO
Bacterial methane oxidation using the enzyme particulate methane monooxygenase (pMMO) contributes to the removal of environmental methane, a potent greenhouse gas. Crystal structures determined using inactive, detergent-solubilized pMMO lack several conserved regions neighboring the proposed active site. We show that reconstituting pMMO in nanodiscs with lipids extracted from the native organism restores methane oxidation activity. Multiple nanodisc-embedded pMMO structures determined by cryo-electron microscopy to 2.14- to 2.46-angstrom resolution reveal the structure of pMMO in a lipid environment. The resulting model includes stabilizing lipids, regions of the PmoA and PmoC subunits not observed in prior structures, and a previously undetected copper-binding site in the PmoC subunit with an adjacent hydrophobic cavity. These structures provide a revised framework for understanding and engineering pMMO function.
Assuntos
Bicamadas Lipídicas , Methylococcus capsulatus/enzimologia , Oxigenases/química , Oxigenases/metabolismo , Domínio Catalítico , Cobre/química , Microscopia Crioeletrônica , Ligação de Hidrogênio , Metano/metabolismo , Modelos Moleculares , Nanoestruturas , Oxirredução , Conformação Proteica , Domínios Proteicos , Subunidades Proteicas/químicaRESUMO
SignificanceMethanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.
Assuntos
Cisteína , Oxazolona , Cobre/metabolismo , Compostos Férricos/química , Humanos , Imidazóis , Oligopeptídeos , Oxigênio/metabolismo , TioamidasRESUMO
The Escherichia coli yobA-yebZ-yebY (AZY) operon encodes the proteins YobA, YebZ, and YebY. YobA and YebZ are homologs of the CopC periplasmic copper-binding protein and the CopD putative copper importer, respectively, whereas YebY belongs to the uncharacterized Domain of Unknown Function 2511 family. Despite numerous studies of E. coli copper homeostasis and the existence of the AZY operon in a range of bacteria, the operon's proteins and their functional roles have not been explored. In this study, we present the first biochemical and functional studies of the AZY proteins. Biochemical characterization and structural modeling indicate that YobA binds a single Cu2+ ion with high affinity. Bioinformatics analysis shows that YebY is widespread and encoded either in AZY operons or in other genetic contexts unrelated to copper homeostasis. We also determined the 1.8 Å resolution crystal structure of E. coli YebY, which closely resembles that of the lantibiotic self-resistance protein MlbQ. Two strictly conserved cysteine residues form a disulfide bond, consistent with the observed periplasmic localization of YebY. Upon treatment with reductants, YebY binds Cu+ and Cu2+ with low affinity, as demonstrated by metal-binding analysis and tryptophan fluorescence. Finally, genetic manipulations show that the AZY operon is not involved in copper tolerance or antioxidant defense. Instead, YebY and YobA are required for the activity of the copper-related NADH dehydrogenase II. These results are consistent with a potential role of the AZY operon in copper delivery to membrane proteins.
Assuntos
Cobre , Proteínas de Escherichia coli , Escherichia coli , Óperon , Proteínas Periplásmicas de Ligação , Quelantes/metabolismo , Cobre/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Relação Estrutura-AtividadeRESUMO
In nature, methane is oxidized to methanol by two enzymes, the iron-dependent soluble methane monooxygenase (sMMO) and the copper-dependent particulate MMO (pMMO). While sMMO's diiron metal active site is spectroscopically and structurally well-characterized, pMMO's copper sites are not. Recent EPR and ENDOR studies have established the presence of two monocopper sites, but the coordination environment of only one has been determined, that within the PmoB subunit and denoted CuB. Moreover, this recent work only focused on a type I methanotrophic pMMO, while previous observations of the type II enzyme were interpreted in terms of the presence of a dicopper site. First, this report shows that the type II Methylocystis species strain Rockwell pMMO, like the type I pMMOs, contains two monocopper sites and that its CuB site has a coordination environment identical to that of type I enzymes. As such, for the full range of pMMOs this report completes the refutation of prior and ongoing suggestions of multicopper sites. Second, and of primary importance, EPR/ENDOR measurements (a) for the first time establish the coordination environment of the spectroscopically observed site, provisionally denoted CuC, in both types of pMMO, thereby (b) establishing the assignment of this site observed by EPR to the crystallographically observed metal-binding site in the PmoC subunit. Finally, these results further indicate that CuC is the likely site of biological methane oxidation by pMMO, a conclusion that will serve as a foundation for proposals regarding the mechanism of this reaction.
Assuntos
Cobre/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Methylocystaceae/enzimologia , Oxigenases/química , Oxigenases/metabolismo , Methylocystaceae/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.
Assuntos
Cobre/metabolismo , Methylosinus/enzimologia , Methylosinus/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/metabolismo , Produtos Biológicos/metabolismo , Quelantes/química , Cobre/química , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Genoma Bacteriano/genética , Imidazóis/metabolismo , Metano/metabolismo , Methylosinus/genética , Methylosinus trichosporium/enzimologia , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Oligopeptídeos/metabolismo , Óperon/genética , Oxirredução , Peptídeos/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Cobre/química , Cinurenina/química , Metaloproteínas/química , Methylosinus trichosporium/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Cristalografia por Raios X , Cinurenina/biossíntese , Cinurenina/genética , Metaloproteínas/genética , Metaloproteínas/metabolismo , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Domínios ProteicosRESUMO
The enzymatic conversion of the greenhouse gas, methane, to a liquid fuel, methanol, is performed by methane monooxygenases (MMOs) under mild conditions. The copper stoichiometry of particulate MMO (pMMO) has been long debated, with a dicopper site previously proposed on the basis of a 2.51 Å Cu-Cu feature in extended X-ray absorption fine structure (EXAFS) data. However, recent crystallographic data and advanced electron paramagnetic resonance (EPR) characterization support the presence of only mononuclear copper sites. To reconcile these data, we have collected high-energy resolution fluorescence detected (HERFD) and partial fluorescence yield (PFY) EXAFS spectra of Methylococcus (M.) capsulatus (Bath) pMMO. Both methods reveal only monocopper sites. These data were compared to previously published pMMO PFY-EXAFS data from M. capsulatus (Bath) and Methylomicrobium alcaliphilum 20Z, supporting dicopper and monocopper sites, respectively. The FT-EXAFS feature previously attributed to a dicopper site can be reproduced by the inclusion of a metallic copper background signal. The exact position of this feature is dependent on the nature of the sample and the percentage of background contamination, indicating that visual inspection is not sufficient for identifying background metallic contributions. Additionally, an undamaged X-ray absorption spectrum was obtained, consistent with the copper oxidation-state speciation determined by EPR quantification. X-ray photodamage studies suggest that the previously observed Cu(i) XAS features are in part attributable to photodamage. This study illustrates the complex array of factors involved in EXAFS measurement and modeling of pMMO and more generally, dilute metalloproteins with multiple metal centers.
RESUMO
Methanotrophic bacteria represent a potential route to methane utilization and mitigation of methane emissions. In the first step of their metabolic pathway, aerobic methanotrophs use methane monooxygenases (MMOs) to activate methane, oxidizing it to methanol. There are two types of MMOs: a particulate, membrane-bound enzyme (pMMO) and a soluble, cytoplasmic enzyme (sMMO). The two MMOs are completely unrelated, with different architectures, metal cofactors, and mechanisms. The more prevalent of the two, pMMO, is copper-dependent, but the identity of its copper active site remains unclear. By contrast, sMMO uses a diiron active site, the catalytic cycle of which is well understood. Here we review the current state of knowledge for both MMOs, with an emphasis on recent developments and emerging hypotheses. In addition, we discuss obstacles to developing expression systems, which are needed to address outstanding questions and to facilitate future protein engineering efforts.
Assuntos
Bactérias/metabolismo , Metano/metabolismo , Oxigenases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Metais/química , Metais/metabolismo , Metano/química , Oxirredução , Oxigenases/química , Oxigenases/genética , Engenharia de ProteínasRESUMO
Copper is critically important for methanotrophic bacteria because their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is copper-dependent. In addition to pMMO, many other copper proteins are encoded in the genomes of methanotrophs, including proteins that contain periplasmic copper-Achaperone (PCuAC) domains. Using bioinformatics analyses, we identified three distinct classes of PCuAC domain-containing proteins in methanotrophs, termed PmoF1, PmoF2, and PmoF3. PCuAC domains from other types of bacteria bind a single Cu(I) ion via an HXnMX21/22HXM motif, which is also present in PmoF3, but PmoF1 and PmoF2 lack this motif entirely. Instead, the PCuAC domains of PmoF1 and PmoF2 bind only Cu(II), and PmoF1 binds additional Cu(II) ions in a His-rich extension to its PCuAC domain. Crystal structures of the PmoF1 and PmoF2 PCuAC domains reveal that Cu(II) is coordinated by an N-terminal histidine brace HX10H motif. This binding site is distinct from those of previously characterized PCuAC domains but resembles copper centers in CopC proteins and lytic polysaccharide monooxygenase (LPMO) enzymes. Bioinformatics analysis of the entire PCuAC family reveals previously unappreciated diversity, including sequences that contain both the HXnMX21/22HXM and HX10H motifs, and sequences that lack either set of copper-binding ligands. These findings provide the first characterization of an additional class of copper proteins from methanotrophs, further expand the PCuAC family, and afford new insight into the biological significance of histidine brace-mediated copper coordination.
Assuntos
Oxigenases/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Sítios de Ligação , Cobre/metabolismo , Cristalografia por Raios X/métodos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Histidina/análogos & derivados , Histidina/química , Histidina/metabolismo , Ligantes , Methylococcaceae/metabolismo , Methylocystaceae/metabolismo , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Compostos Organometálicos/metabolismo , Domínios ProteicosRESUMO
Methanobactins (Mbns) are ribosomally-produced, post-translationally modified peptidic copper-binding natural products produced under conditions of copper limitation. Genes encoding Mbn biosynthetic and transport proteins have been identified in a wide variety of bacteria, indicating a broader role for Mbns in bacterial metal homeostasis. Many of the genes in the Mbn operons have been assigned functions, but two genes usually present, mbnP and mbnH, encode uncharacterized proteins predicted to reside in the periplasm. MbnH belongs to the bacterial diheme cytochrome c peroxidase (bCcP)/MauG protein family, and MbnP contains no domains of known function. Here, we performed a detailed bioinformatic analysis of both proteins and have biochemically characterized MbnH from Methylosinus (Ms.) trichosporium OB3b. We note that the mbnH and mbnP genes typically co-occur and are located proximal to genes associated with microbial copper homeostasis. Our bioinformatics analysis also revealed that the bCcP/MauG family is significantly more diverse than originally appreciated, and that MbnH is most closely related to the MauG subfamily. A 2.6 Å resolution structure of Ms. trichosporium OB3b MbnH combined with spectroscopic data and peroxidase activity assays provided evidence that MbnH indeed more closely resembles MauG than bCcPs, although its redox properties are significantly different from those of MauG. The overall similarity of MbnH to MauG suggests that MbnH could post-translationally modify a macromolecule, such as internalized CuMbn or its uncharacterized partner protein, MbnP. Our results indicate that MbnH is a MauG-like diheme protein that is likely involved in microbial copper homeostasis and represents a new family within the bCcP/MauG superfamily.
Assuntos
Cobre/metabolismo , Imidazóis/metabolismo , Methylosinus trichosporium/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Sequência de Aminoácidos/genética , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Homeostase , Oligopeptídeos/biossíntese , Óperon/genética , Processamento de Proteína Pós-TraducionalRESUMO
Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes.