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.

Biosynthesis of chiral diols from alkenes using metabolically engineered type II methanotroph.

Methanotrophs are environmentally friendly microorganisms capable of converting gas to liquid using methane monooxygenases (MMOs). In addition to methane-to-methanol conversion, MMOs catalyze the conversion of alkanes to alcohols and alkenes to epoxides. Herein, the efficacy of epoxidation by type I and II methanotrophs was investigated, and type II methanotrophs were observed to be more efficient in converting alkenes to epoxides. Subsequently, three (Epoxide hydrolase) EHs of different origins were overexpressed in the type II methanotroph Methylosinus trichosporium OB3b to produce 1,2-diols from epoxide. Methylosinus trichosporium OB3b expressing Caulobacter crescentus EH produced the highest amount of (R)-1,2-propanediol (251.5 mg/L) from 1-propene. These results demonstrate the possibility of using methanotrophs as a microbial platform for diol production and the development of a continuous bioreactor for industrial applications.

Inhibition of nitrous oxide reduction in forest soil microcosms by different forms of methanobactin.

Copper plays a critical role in controlling greenhouse gas emissions as it is a key component of the particulate methane monooxygenase and nitrous oxide reductase. Some methanotrophs excrete methanobactin (MB) that has an extremely high copper affinity. As a result, MB may limit the ability of other microbes to gather copper, thereby decreasing their activity as well as impacting microbial community composition. Here, we show using forest soil microcosms that multiple forms of MB; MB from Methylosinus trichosporium OB3b (MB-OB3b) and MB from Methylocystis sp. strain SB2 (MB-SB2) increased nitrous oxide (N2 O) production as well caused significant shifts in microbial community composition. Such effects, however, were mediated by the amount of copper in the soils, with low-copper soil microcosms showing the strongest response to MB. Furthermore, MB-SB2 had a stronger effect, likely due to its higher affinity for copper. The presence of either form of MB also inhibited nitrite reduction and generally increased the presence of genes encoding for the iron-containing nitrite reductase (nirS) over the copper-dependent nitrite reductase (nirK). These data indicate the methanotrophic-mediated production of MB can significantly impact multiple steps of denitrification, as well as have broad effects on microbial community composition of forest soils.

Important Structural Features of Thiolate-Rich Four-Helix Bundles for Cu(I) Uptake and Removal.

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.

Capturing a bis-Fe(IV) State in Methylosinus trichosporium OB3b MbnH.

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.

Identification of a TonB-Dependent Receptor Involved in Lanthanide Switch by the Characterization of Laboratory-Adapted Methylosinus trichosporium OB3b.

Two methanol dehydrogenases (MDHs), MxaFI and XoxF, have been characterized in methylotrophic and methanotrophic bacteria. MxaFI contains a calcium ion in its active site, whereas XoxF contains a lanthanide ion. Importantly, the expression of MxaFI and XoxF is inversely regulated by lanthanide bioavailability, i.e., the "lanthanide switch." To reveal the genetic and environmental factors affecting the lanthanide switch, we focused on two Methylosinus trichosporium OB3b mutants isolated during routine cultivation. In these mutants, MxaF was constitutively expressed, but lanthanide-dependent XoxF1 was not, even in the presence of 25 µM cerium ions, which is sufficient for XoxF expression in the wild type. Genotyping showed that both mutants harbored a loss-of-function mutation in the CQW49_RS02145 gene, which encodes a TonB-dependent receptor. Gene disruption and complementation experiments demonstrated that CQW49_RS02145 was required for XoxF1 expression in the presence of 25 µM cerium ions. Phylogenetic analysis indicated that CQW49_RS02145 was homologous to the Methylorubrum extorquens AM1 lanthanide transporter gene (lutH). These findings suggest that CQW49_RS02145 is involved in lanthanide uptake across the outer membrane. Furthermore, we demonstrated that supplementation with cerium and glycerol caused severe growth arrest in the wild type. CQW49_RS02145 underwent adaptive laboratory evolution in the presence of cerium and glycerol ions, resulting in a mutation that partially mitigated the growth arrest. This finding implies that loss-of-function mutations in CQW49_RS02145 can be attributed to residual glycerol from the frozen stock. IMPORTANCE Lanthanides are widely used in many industrial applications, including catalysts, magnets, and polishing. Recently, lanthanide-dependent metabolism was characterized in methane-utilizing bacteria. Despite the global demand for lanthanides, few studies have investigated the mechanism of lanthanide uptake by these bacteria. In this study, we identify a lanthanide transporter in Methylosinus trichosporium OB3b and indicate the potential interaction between intracellular lanthanide and glycerol. Understanding the genetic and environmental factors affecting lanthanide uptake should not only help improve the use of lanthanides for the bioconversion of methane into valuable products like methanol but also be of value for developing biomining to extract lanthanides under neutral conditions.

Process engineering strategy for improved methanol production in Methylosinus trichosporium through enhanced mass transfer and solubility of methane and carbon dioxide.

Methanol was produced in a two-stage integrated process using Methylosinus trichosporium NCIMB 11131. The first stage involved sequestration of methane to produce methanotrophic biomass, which was utilized as biocatalyst in the second stage to convert CO2 into methanol. A combinatorial process engineering approach of design of micro-sparger, engagement of draft tube, addition of mass transfer vector and elevation of reactor operating pressure was employed to enhance production of biomass and methanol. Maximum biomass titer of 7.68 g/L and productivity of 1.46 g/L d-1 were achieved in an airlift reactor equipped with a micro-sparger of 5 µm pore size, in the presence of draft tube and 10 % v/v silicone oil, as mass transfer vector. Maximum methane fixation rate was estimated to be 0.80 g/L d-1. Maximum methanol titer of 1.98 g/L was achieved under an elevated operating pressure of 4 bar in a high-pressure stirred tank reactor.

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.

The Proteobacterial Methanotroph Methylosinus trichosporium OB3b Remodels Membrane Lipids in Response to Phosphate Limitation.

Methane is a potent greenhouse gas in the atmosphere, and its concentration has continued to increase in recent decades. Aerobic methanotrophs, bacteria that use methane as the sole carbon source, are an important biological sink for methane, and they are widely distributed in the natural environment. However, relatively little is known on how methanotroph activity is regulated by nutrients, particularly phosphorus (P). P is the principal nutrient constraining plant and microbial productivity in many ecosystems, ranging from agricultural land to the open ocean. Using a model methanotrophic bacterium, Methylosinus trichosporium OB3b, we demonstrate here that this bacterium can produce P-free glycolipids to replace membrane phospholipids in response to P limitation. The formation of the glycolipid monoglucuronic acid diacylglycerol requires plcP-agt genes since the plcP-agt mutant is unable to produce this glycolipid. This plcP-agt-mediated lipid remodeling pathway appears to be important for M. trichosporium OB3b to cope with P stress, and the mutant grew significantly slower under P limitation. Interestingly, comparative genomics analysis shows that the ability to perform lipid remodeling appears to be a conserved trait in proteobacterial methanotrophs; indeed, plcP is found in all proteobacterial methanotroph genomes, and plcP transcripts from methanotrophs are readily detectable in metatranscriptomics data sets. Together, our study provides new insights into the adaptation to P limitation in this ecologically important group of bacteria. IMPORTANCE Methane is a potent greenhouse gas in the atmosphere, and its concentration has continued to increase steadily in recent decades. In the natural environment, bacteria known as methanotrophs help mitigate methane emissions at no cost to human beings. However, relatively little is known regarding how methane oxidation activity in methanotrophs is regulated by soil nutrients, particularly phosphorus. Here, we show that methanotrophs can modify their membrane in response to phosphorus limitation and that the ability to change membrane lipids is important for methanotroph activity. Genome and metatranscriptome analyses suggest that such an adaptation strategy appears to be strictly conserved in all proteobacterial methanotrophs and is used by these bacteria in the natural environment. Together, our study provides a plausible molecular mechanism for better understanding the role of phosphorus on methane oxidation in the natural environment.

Enhancement of Methane Catalysis Rates in Methylosinus trichosporium OB3b.

Particulate methane monooxygenase (pMMO), a membrane-bound enzyme having three subunits (α, ß, and γ) and copper-containing centers, is found in most of the methanotrophs that selectively catalyze the oxidation of methane into methanol. Active sites in the pMMO of Methylosinus trichosporium OB3b were determined by docking the modeled structure with ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene. The docking energy between the modeled pMMO structure and ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene was -5.2, -5.7, -4.2, and -3.8 kcal/mol, respectively, suggesting the existence of more than one active site within the monomeric subunits due to the presence of multiple binding sites within the pMMO monomer. The evaluation of tunnels and cavities of the active sites and the docking results showed that each active site is specific to the radius of the substrate. To increase the catalysis rates of methane in the pMMO of M. trichosporium OB3b, selected amino acid residues interacting at the binding site of ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene were mutated. Based on screening the strain energy, docking energy, and physiochemical properties, five mutants were downselected, B:Leu31Ser, B:Phe96Gly, B:Phe92Thr, B:Trp106Ala, and B:Tyr110Phe, which showed the docking energy of -6.3, -6.7, -6.3, -6.5, and -6.5 kcal/mol, respectively, as compared to the wild type (-5.2 kcal/mol) with ethylbenzene. These results suggest that these five mutants would likely increase methane oxidation rates compared to wild-type pMMO.

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.

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.

Two-stage integrated process for bio-methanol production coupled with methane and carbon dioxide sequestration: Kinetic modelling and experimental validation.

The study demonstrates a two-stage integrated process for bio-methanol production using Methylosinus trichosporium NCIMB 11131, coupled with sequestration of methane and carbon dioxide. The first stage involved generation of methanotrophic biomass via sequestration of methane; which was used as biocatalyst to reduce carbon dioxide into methanol in the second stage. Maximum biomass titer of 3.39 g L-1 and productivity of 0.60 g L-1 d-1 were achieved in semi-batch stirred tank reactor with methane concentration in the inlet gas mixture of 2.5% v/v and gas flow rate of 0.5 vvm. Methane fixation rate was estimated to be 0.32 g L-1 d-1. Maximum methanol titer of 0.58 g L-1 was achieved at headspace carbon dioxide concentration of 50% v/v and liquid to headspace volume ratio 10:90. Subsequently, a kinetic model was developed to predict and understand the system behaviour in terms of dynamic profile of growth, methanol formation, concentration of dissolved methane or carbon dioxide in the aqueous phase and headspace carbon dioxide concentration, in response to varying process parameters. The model can serve as a tool for estimation of process parameters and aid in overall production optimization.

X-ray Crystal Structures of Methane Monooxygenase Hydroxylase Complexes with Variants of Its Regulatory Component: Correlations with Altered Reaction Cycle Dynamics.

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.

Evidence for methanobactin "Theft" and novel chalkophore production in methanotrophs: impact on methanotrophic-mediated methylmercury degradation.

Aerobic methanotrophy is strongly controlled by copper, and methanotrophs are known to use different mechanisms for copper uptake. Some methanotrophs secrete a modified polypeptide-methanobactin-while others utilize a surface-bound protein (MopE) and a secreted form of it (MopE*) for copper collection. As different methanotrophs have different means of sequestering copper, competition for copper significantly impacts methanotrophic activity. Herein, we show that Methylomicrobium album BG8, Methylocystis sp. strain Rockwell, and Methylococcus capsulatus Bath, all lacking genes for methanobactin biosynthesis, are not limited for copper by multiple forms of methanobactin. Interestingly, Mm. album BG8 and Methylocystis sp. strain Rockwell were found to have genes similar to mbnT that encodes for a TonB-dependent transporter required for methanobactin uptake. Data indicate that these methanotrophs "steal" methanobactin and such "theft" enhances the ability of these strains to degrade methylmercury, a potent neurotoxin. Further, when mbnT was deleted in Mm. album BG8, methylmercury degradation in the presence of methanobactin was indistinguishable from when MB was not added. Mc. capsulatus Bath lacks anything similar to mbnT and was unable to degrade methylmercury either in the presence or absence of methanobactin. Rather, Mc. capsulatus Bath appears to rely on MopE/MopE* for copper collection. Finally, not only does Mm. album BG8 steal methanobactin, it synthesizes a novel chalkophore, suggesting that some methanotrophs utilize both competition and cheating strategies for copper collection. Through a better understanding of these strategies, methanotrophic communities may be more effectively manipulated to reduce methane emissions and also enhance mercury detoxification in situ.

Characterization of a Copper-Chelating Natural Product from the Methanotroph Methylosinus sp. LW3.

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.

Copper binding by a unique family of metalloproteins is dependent on kynurenine formation.

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 ∼2.9 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.

Soluble Methane Monooxygenase Component Interactions Monitored by 19F NMR.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the fissure of the C-H bond of methane and the insertion of one atom of oxygen from O2 to yield methanol. Efficient multiple-turnover catalysis occurs only in the presence of all three sMMO protein components: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB). The complex series of sMMO protein component interactions that regulate the formation and decay of sMMO reaction cycle intermediates is not fully understood. Here, the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is 19F-labeled by reaction of the K15C variant with 3-bromo-1,1,1-trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation, allowing detailed studies of the interactions with sMMOH using 19F NMR. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFA-K15C-5FW-MMOB are readily detected at 5 µM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar KD values, independent of the oxidation state of the sMMOH diiron cluster. These findings suggest a new model for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate Q before it can react with methane.