Heterologous Biosynthesis of Methanobactin from Methylocystis sp. Strain SB2 in Methylosinus trichosporium OB3b.

Aerobic methanotrophs, or methane-consuming microbes, are strongly dependent on copper for their activity. To satisfy this requirement, some methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin (MB). In addition to playing a critical role in methanotrophy, MB has also been shown to have great promise in treating copper-related human diseases, perhaps most significantly Wilson's disease. In this congenital disorder, copper builds up in the liver, leading to irreversible damage and, in severe cases, complete organ failure. Remarkably, MB has been shown to reverse such damage in animal models, and there is a great deal of interest in upscaling MB production for expanded clinical trials. Such efforts, however, are currently hampered as (1) the natural rate of MB production rate by methanotrophs is low, (2) the use of methane as a substrate for MB production is problematic as it is explosive in air, (3) there is limited understanding of the entire pathway of MB biosynthesis, and (4) the most attractive form of MB is produced by Methylocystis sp. strain SB2, a methanotroph that is genetically intractable. Herein, we report heterologous biosynthesis of MB from Methylocystis sp. strain SB2 in an alternative methanotroph, Methylosinus trichosporium OB3b, not only on methane but also on methanol. As a result, the strategy described herein not only facilitates enhanced MB production but also provides opportunities to construct various mutants to delineate the entire pathway of MB biosynthesis, as well as the creation of modified forms of MB that may have enhanced therapeutic value.

Copper impairs the intestinal barrier integrity in Wilson disease.

In Wilson disease (WD), liver copper (Cu) excess, caused by mutations in the ATPase Cu transporting beta (ATP7B), has been extensively studied. In contrast, in the gastrointestinal tract, responsible for dietary Cu uptake, ATP7B malfunction is poorly explored. We therefore investigated gut biopsies from WD patients and compared intestines from two rodent WD models and from human ATP7B knock-out intestinal cells to their respective wild-type controls. We observed gastrointestinal (GI) inflammation in patients, rats and mice lacking ATP7B. Mitochondrial alterations and increased intestinal leakage were observed in WD rats, Atp7b-/- mice and human ATP7B KO Caco-2 cells. Proteome analyses of intestinal WD homogenates revealed profound alterations of energy and lipid metabolism. The intestinal damage in WD animals and human ATP7B KO cells did not correlate with absolute Cu elevations, but likely reflects intracellular Cu mislocalization. Importantly, Cu depletion by the high-affinity Cu chelator methanobactin (MB) restored enterocyte mitochondria, epithelial integrity, and resolved gut inflammation in WD rats and human WD enterocytes, plausibly via autophagy-related mechanisms. Thus, we report here before largely unrecognized intestinal damage in WD, occurring early on and comprising metabolic and structural tissue damage, mitochondrial dysfunction, and compromised intestinal barrier integrity and inflammation, that can be resolved by high-affinity Cu chelation treatment.

Study on the Relationship between Particulate Methane Monooxygenase and Methanobactin on Gold-Nanoparticles-Modified Electrodes.

(1) Background: Particulate methane monooxygenase (pMMO) has a strong dependence on the natural electron transfer path and is prone to denaturation, which results in its redox activity centers being unable to transfer electrons with bare electrodes directly and making it challenging to observe an electrochemical response; (2) Methods: Using methanobactin (Mb) as the electron transporter between gold electrodes and pMMO, a bionic interface with high biocompatibility and stability was created. The Mb-AuNPs-modified functionalized gold net electrode as a working electrode, the kinetic behaviors of pMMO bioelectrocatalysis, and the effect of Mb on pMMO were analyzed. The CV tests were performed at different scanning rates to obtain electrochemical kinetics parameters. (3) Results: The values of the electron transfer coefficient (α) and electron transfer rate constant (ks) are relatively large in test environments containing only CH4 or O2. In contrast, in the test environment containing both CH4 and O2, the bioelectrocatalysis of pMMO is a two-electron transfer process with a relatively small α and ks; (4) Conclusions: It was inferred that Mb formed the complex with pMMO. More importantly, Mb not only played a role in electron transfer but also in stabilizing the enzyme structure of pMMO and maintaining a specific redox state. Furthermore, the continuous catalytic oxidation of natural substrate methane was realized.

Enhanced triolein and ethyl ferulate interesterification performance by CRL-AuNPs.

This study established a Candida rugosa lipase (CRL) system to catalyze triolein and ethyl ferulate interesterification. The products were identified, and the binding mode between the substrates and CRL was predicted through molecular docking. Three methods for preparing CRL-AuNPs were proposed and characterized. It was found that the addition of 40 mL of 15 nm gold nanoparticles increased the CRL activity from 3.05 U/mg to 4.75 U/mg, but the hybridization efficiency was only 32.7 %. By using 4 mL of 0.1 mg/mL chloroauric acid, the hybridization efficiency was improved to 50.7 %, but the enzyme activity was sharply decreased. However, when the molar ratio of Mb to HAuCl4 was 0.2, the hybridization efficiency increased to 71.8 %, and the CRL activity was also enhanced to 5.98 U/mg. Under optimal conditions, the enzyme activity of CRL-AuNPs③ was maintained at 95 % after 6 repetitions and 85.6 % after 30 days at room temperature.

MmoD regulates soluble methane monooxygenase and methanobactin production in Methylosinus trichosporium OB3b.

IMPORTANCE: Aerobic methanotrophs play a critical role in the global carbon cycle, particularly in controlling net emissions of methane to the atmosphere. As methane is a much more potent greenhouse gas than carbon dioxide, there is increasing interest in utilizing these microbes to mitigate future climate change by increasing their ability to consume methane. Any such efforts, however, require a detailed understanding of how to manipulate methanotrophic activity. Herein, we show that methanotrophic activity is strongly controlled by MmoD, i.e., MmoD regulates methanotrophy through the post-transcriptional regulation of the soluble methane monooxygenase and controls the ability of methanotrophs to collect copper. Such data are likely to prove quite useful in future strategies to enhance the use of methanotrophs to not only reduce methane emissions but also remove methane from the atmosphere.

ARBM101 (Methanobactin SB2) Drains Excess Liver Copper via Biliary Excretion in Wilson's Disease Rats.

BACKGROUND & AIMS: Excess copper causes hepatocyte death in hereditary Wilson's disease (WD). Current WD treatments by copper-binding chelators may gradually reduce copper overload; they fail, however, to bring hepatic copper close to normal physiological levels. Consequently, lifelong daily dose regimens are required to hinder disease progression. This may result in severe issues due to nonadherence or unwanted adverse drug reactions and also due to drug switching and ultimate treatment failures. This study comparatively tested bacteria-derived copper binding agents-methanobactins (MBs)-for efficient liver copper depletion in WD rats as well as their safety and effect duration. METHODS: Copper chelators were tested in vitro and in vivo in WD rats. Metabolic cage housing allowed the accurate assessment of animal copper balances and long-term experiments related to the determination of minimal treatment phases. RESULTS: We found that copper-binding ARBM101 (previously known as MB-SB2) depletes WD rat liver copper dose dependently via fecal excretion down to normal physiological levels within 8 days, superseding the need for continuous treatment. Consequently, we developed a new treatment consisting of repetitive cycles, each of ∼1 week of ARBM101 applications, followed by months of in-between treatment pauses to ensure a healthy long-term survival in WD rats. CONCLUSIONS: ARBM101 safely and efficiently depletes excess liver copper from WD rats, thus allowing for short treatment periods as well as prolonged in-between rest periods.

A turn-on fluorescent nano-probe base on methanobactin-AuNPs for simple and efficient detection of nitrite.

Nitrite ions are important markers threatening humans and environmental security. A highly selective method for rapid detection of nitrite needs to be developed. Herein, a novel and rapid fluorescence method for nitrite determination is established on the basis of diazotization-coupling reaction of methanobactin (Mb) extracted by Methylosinus trichosporium OB3b with nitrite on the fluorescence. In the presence of gold nanoparticles (AuNPs), the fluorescence of AuNPs was strongly quenched by the Mb because the sulfhydryl or amino structures on the surface of Mb could be bound to the surface of AuNPs by forming Au-S or Au-N bonds. Upon addition of nitrite, the Mb easily reacts with nitrite to form azo products in the acidic medium. Then, with the increase of nitrite concentration, the Mb-AuNPs fluorescence was gradually recovered, realizing the turn-on fluorescence sensing of nitrite. Under optimal conditions, the proposed method has a good linear relationship with nitrite concentration in the range of 0-8.0 µM and 8.0-50.0 µM, and the detection limit is 16.21 nM. In addition, satisfactory results were obtained for nitrite analysis using milk, ham sausage and leaf mustard as real samples, which demonstrated that the method as-developed would have great practical application prospects.

Ecological Aerobic Ammonia and Methane Oxidation Involved Key Metal Compounds, Fe and Cu.

Interactions between metals and microbes are critical in geomicrobiology and vital in microbial ecophysiological processes. Methane-oxidizing bacteria (MOB) and ammonia-oxidizing microorganisms (AOM) are key members in aerobic environments to start the C and N cycles. Ammonia and methane are firstly oxidized by copper-binding metalloproteins, monooxygenases, and diverse iron and copper-containing enzymes that contribute to electron transportation in the energy gain pathway, which is evolutionally connected between MOB and AOM. In this review, we summarized recently updated insight into the diverse physiological pathway of aerobic ammonia and methane oxidation of different MOB and AOM groups and compared the metabolic diversity mediated by different metalloenzymes. The elevation of iron and copper concentrations in ecosystems would be critical in the activity and growth of MOB and AOM, the outcome of which can eventually influence the global C and N cycles. Therefore, we also described the impact of various concentrations of metal compounds on the physiology of MOB and AOM. This review study could give a fundamental strategy to control MOB and AOM in diverse ecosystems because they are significantly related to climate change, eutrophication, and the remediation of contaminated sites for detoxifying pollutants.

Multiple Mechanisms for Copper Uptake by Methylosinus trichosporium OB3b in the Presence of Heterologous Methanobactin.

Methanotrophs require copper for their activity as it plays a critical role in the oxidation of methane to methanol. To sequester copper, some methanotrophs secrete a copper-binding compound termed methanobactin (MB). MB, after binding copper, is reinternalized via a specific outer membrane TonB-dependent transporter (TBDT). Methylosinus trichosporium OB3b has two such TBDTs (MbnT1 and MbnT2) that enable M. trichosporium OB3b to take up not only its own MB (MB-OB3b) but also heterologous MB produced from other methanotrophs, e.g., MB of Methylocystis sp. strain SB2 (MB-SB2). Here, we show that uptake of copper in the presence of heterologous MB-SB2 can either be achieved by initiating transcription of mbnT2 or by using its own MB-OB3b to extract copper from MB-SB2. Transcription of mbnT2 is mediated by the N-terminal signaling domain of MbnT2 together with an extracytoplasmic function sigma factor and an anti-sigma factor encoded by mbnI2 and mbnR2, respectively. Deletion of mbnI2R2 or excision of the N-terminal region of MbnT2 abolished induction of mbnT2. However, copper uptake from MB-SB2 was still observed in M. trichosporium OB3b mutants that were defective in MbnT2 induction/function, suggesting another mechanism for uptake copper-loaded MB-SB2. Additional deletion of MB-OB3b synthesis genes in the M. trichosporium OB3b mutants defective in MbnT2 induction/function disrupted their ability to take up copper in the presence of MB-SB2, indicating a role of MB-OB3b in copper extraction from MB-SB2. IMPORTANCE Methanotrophs play a critical role in the global carbon cycle, as well as in future strategies for mitigating climate change through their consumption of methane, a trace atmospheric gas much more potent than carbon dioxide in global warming potential. Copper uptake is critical for methanotrophic activity, and here, we show different approaches for copper uptake. This study expands our knowledge and understanding of how methanotrophs collect and compete for copper, and such information may be useful in future manipulation of methanotrophs for a variety of environmental and industrial applications.

Copper mobilisation from Cu sulphide minerals by methanobactin: Effect of pH, oxygen and natural organic matter.

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.

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis.

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.

Variable Inhibition of Nitrous Oxide Reduction in Denitrifying Bacteria by Different Forms of Methanobactin.

Aerobic methanotrophic activity is highly dependent on copper availability, and methanotrophs have developed multiple strategies to collect copper. Specifically, when copper is limiting (ambient concentrations less than 1 µM), some methanotrophs produce and secret a small modified peptide (less than 1,300 Da) termed methanobactin (MB) that binds copper with high affinity. As MB is secreted into the environment, other microbes that require copper for their metabolism may be inhibited as MB may make copper unavailable; e.g., inhibition of denitrifiers as complete conversion nitrate to dinitrogen involves multiple enzymes, some of which are copper-dependent. Of key concern is inhibition of the copper-dependent nitrous oxide reductase (NosZ), the only known enzyme capable of converting nitrous oxide (N2O) to dinitrogen. Herein, we show that different forms of MB differentially affect copper uptake and N2O reduction by Pseudomonas stutzeri strain DCP-Ps1 (that expresses clade I NosZ) and Dechloromonas aromatica strain RCB (that expresses clade II NosZ). Specifically, in the presence of MB from Methylocystis sp. strain SB2 (SB2-MB), copper uptake and nosZ expression were more significantly reduced than in the presence of MB from Methylosinus trichosporium OB3b (OB3b-MB). Further, N2O accumulation increased more significantly for both P. stutzeri strain DCP-Ps1 and D. aromatica strain RCB in the presence of SB2-MB versus OB3b-MB. These data illustrate that copper competition between methanotrophs and denitrifying bacteria can be significant and that the extent of such competition is dependent on the form of MB that methanotrophs produce. IMPORTANCE Herein, it was demonstrated that the different forms of methanobactin differentially enhance N2O emissions from Pseudomonas stutzeri strain DCP-Ps1 (harboring clade I nitrous oxide reductase) and Dechloromonas aromatica strain RCB (harboring clade II nitrous oxide reductase). This work contributes to our understanding of how aerobic methanotrophs compete with denitrifiers for the copper uptake and also suggests how MBs prevent copper collection by denitrifiers, thus downregulating expression of nitrous oxide reductase. This study provides critical information for enhanced understanding of microbe-microbe interactions that are important for the development of better predictive models of net greenhouse gas emissions (i.e., methane and nitrous oxide) that are significantly controlled by microbial activity.

Simple and rapid detection of calcium peroxide in flour based on methanobactin peroxidase-like activity.

Calcium peroxide is forbidden to be added to flour as brightener in many countries. A rapid and sensitive spectrophotometric method for the detection of calcium peroxide in flour was proposed. Methanobactin (Mb), a copper-binding small peptide of methanotrophs with excellent peroxidase-like activity, has been successfully applied for H2O2-mediated co-oxidation between phenol and 4-aminoantipyrine to give a colored product that can be detected at 505 nm. When the concentration of Mb-Cu was 6.5 × 10-6 mol/L, the detection temperature was 50 ℃, and the detection time was 5 min, the linear range for quantification of calcium peroxide concentration was observed between 0.4 and 10.0 mg/L with R2 = 0.99397. The limit of detection was 0.027 mg/L (3.34 mg/kg flour) and the average recovery of standard addition was 99.6%-100.5%. Mb was very stable and still maintained catalytic activity even at 50 ℃ in acidic medium, which gave the proposed method has simple process and rapid detection speed.

Two TonB-Dependent Transporters in Methylosinus trichosporium OB3b Are Responsible for Uptake of Different Forms of Methanobactin and Are Involved in the Canonical "Copper Switch".

Copper is an important component of methanotrophic physiology, as it controls the expression and activity of alternative forms of methane monooxygenase (MMO). To collect copper, some methanotrophs secrete a chalkophore- or copper-binding compound called methanobactin (MB). MB is a ribosomally synthesized posttranslationally modified polypeptide (RiPP) that, after binding copper, is collected by MbnT, a TonB-dependent transporter (TBDT). Structurally different forms of MB have been characterized, and here, we show that different forms of MB are collected by specific TBDTs. Further, we report that in the model methanotroph, Methylosinus trichosporium OB3b, expression of the TBDT required for uptake of a different MB made by Methylocystis sp. strain SB2 (MB-SB2) is induced in the presence of MB-SB2, suggesting that methanotrophs have developed specific machinery and regulatory systems to actively take up MB from other methanotrophs for copper collection. Moreover, the canonical "copper switch" in M. trichosporium OB3b that controls expression of alternative MMOs is apparent if one of the two TBDTs required for MB-OB3b and MB-SB2 uptake is knocked out, but is disrupted if both TBDTs are knocked out. These data indicate that MB uptake, including the uptake of exogenous MB, plays an important role in the copper switch in M. trichosporium OB3b and, thus, overall activity. Based on these data, we propose a revised model for the copper switch in this methanotroph that involves MB uptake. IMPORTANCE In this study, we demonstrate that different TBDTs in the model methanotroph Methylosinus trichosporium OB3b are responsible for uptake of either endogenous MB or exogenous MB. Interestingly, the presence of exogenous MB induces expression of its specific TBDT in M. trichosporium OB3b, suggesting that this methanotroph is able to actively take up MB produced by others. This work contributes to our understanding of how microbes collect and compete for copper and also helps inform how such uptake coordinates the expression of different forms of methane monooxygenase. Such studies are likely to be very important to develop a better understanding of methanotrophic interactions via synthesis and secretion of secondary metabolites such as methanobactin and thus provide additional means whereby these microbes can be manipulated for a variety of environmental and industrial purposes.

MbnC Is Not Required for the Formation of the N-Terminal Oxazolone in the Methanobactin from Methylosinus trichosporium OB3b.

Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b is mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons and, in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the ΔmbnC mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals and nanoparticle formation, as well as the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for posttranslational modification(s) of the two oxazolone groups are not identical.

Biomedical Applications of Biomolecules Isolated from Methanotrophic Bacteria in Wastewater Treatment Systems.

Wastewater treatment plants and other remediation facilities serve important roles, both in public health, but also as dynamic research platforms for acquiring useful resources and biomolecules for various applications. An example of this is methanotrophic bacteria within anaerobic digestion processes in wastewater treatment plants. These bacteria are an important microbial source of many products including ectoine, polyhydroxyalkanoates, and methanobactins, which are invaluable to the fields of biotechnology and biomedicine. Here we provide an overview of the methanotrophs' unique metabolism and the biochemical pathways involved in biomolecule formation. We also discuss the potential biomedical applications of these biomolecules through creation of beneficial biocompatible products including vaccines, prosthetics, electronic devices, drug carriers, and heart stents. We highlight the links between molecular biology, public health, and environmental science in the advancement of biomedical research and industrial applications using methanotrophic bacteria in wastewater treatment systems.

Metal(loid) speciation and transformation by aerobic methanotrophs.

Manufacturing and resource industries are the key drivers for economic growth with a huge environmental cost (e.g. discharge of industrial effluents and post-mining substrates). Pollutants from waste streams, either organic or inorganic (e.g. heavy metals), are prone to interact with their physical environment that not only affects the ecosystem health but also the livelihood of local communities. Unlike organic pollutants, heavy metals or trace metals (e.g. chromium, mercury) are non-biodegradable, bioaccumulate through food-web interactions and are likely to have a long-term impact on ecosystem health. Microorganisms provide varied ecosystem services including climate regulation, purification of groundwater, rehabilitation of contaminated sites by detoxifying pollutants. Recent studies have highlighted the potential of methanotrophs, a group of bacteria that can use methane as a sole carbon and energy source, to transform toxic metal (loids) such as chromium, mercury and selenium. In this review, we synthesise recent advances in the role of essential metals (e.g. copper) for methanotroph activity, uptake mechanisms alongside their potential to transform toxic heavy metal (loids). Case studies are presented on chromium, selenium and mercury pollution from the tanneries, coal burning and artisanal gold mining, respectively, which are particular problems in the developing economy that we propose may be suitable for remediation by methanotrophs. Video Abstract.

Spectroscopic and computational investigations of organometallic complexation of group 12 transition metals by methanobactins from Methylocystis sp. SB2.

Methanotrophic bacteria catalyze the aerobic oxidation of methane to methanol using Cu-containing enzymes, thereby exerting a modulating influence on the global methane cycle. To facilitate the acquisition of Cu ions, some methanotrophic bacteria secrete small modified peptides known as "methanobactins," which strongly bind Cu and function as an extracellular Cu recruitment relay, analogous to siderophores and Fe. In addition to Cu, methanobactins form complexes with other late transition metals, including the Group 12 transition metals Zn, Cd, and Hg, although the interplay among solution-phase configurations, metal interactions, and the spectroscopic signatures of methanobactin-metal complexes remains ambiguous. In this study, the complexation of Zn, Cd, and Hg by methanobactin from Methylocystis sp. strain SB2 was studied using a combination of absorbance, fluorescence, extended x-ray absorption fine structure (EXAFS) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. We report changes in sample absorbance and fluorescence spectral dynamics, which occur on a wide range of experimental timescales and characterize a clear stoichiometric complexation dependence. Mercury L3-edge EXAFS and TD-DFT calculations suggest a linear model for HgS coordination, and TD-DFT suggests a tetrahedral model for Zn2+ and Cd2+. We observed an enhancement in the fluorescence of methanobactin upon interaction with transition metals and propose a mechanism of complexation-hindered isomerization drawing inspiration from the wild-type Green Fluorescent Protein active site. Collectively, our results represent the first combined computational and experimental spectroscopy study of methanobactins and shed new light on molecular interactions and dynamics that characterize complexes of methanobactins with Group 12 transition metals.

The enzymology of oxazolone and thioamide synthesis in methanobactin.

Methanobactins are ribosomally synthesized and post-translationally modified peptidic (RiPP) natural products that are known for their ability to chelate copper ions. Crucial for their high copper affinity is a pair of bidentate ligands comprising a nitrogen-containing heterocycle and an adjacent thioamide or enethiol group. The previously uncharacterized proteins MbnB and MbnC were recently shown to synthesize these groups. In this chapter, we describe the methods that were used to determine that MbnB and MbnC are the core biosynthetic enzymes in methanobactin biosynthesis. The two proteins form a heterodimeric complex (MbnBC) which, through a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA), modifies a cysteine residue in order to install the oxazolone and thioamide moieties. This overview covers the heterologous expression and purification of MbnBC, characterization of the iron cluster found in MbnB, and characterization of the modification installed on MbnA. While this chapter is specific to MbnBC, the methods outlined here can be broadly applied to the enzymology of other proteins that install similar groups as well as enzyme pairs related to MbnB and MbnC.

Oxygen Generation via Water Splitting by a Novel Biogenic Metal Ion-Binding Compound.

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.