Chalkophores.

Copper-binding metallophores, or chalkophores, play a role in microbial copper homeostasis that is analogous to that of siderophores in iron homeostasis. The best-studied chalkophores are members of the methanobactin (Mbn) family-ribosomally produced, posttranslationally modified natural products first identified as copper chelators responsible for copper uptake in methane-oxidizing bacteria. To date, Mbns have been characterized exclusively in those species, but there is genomic evidence for their production in a much wider range of bacteria. This review addresses the current state of knowledge regarding the function, biosynthesis, transport, and regulation of Mbns. While the roles of several proteins in these processes are supported by substantial genetic and biochemical evidence, key aspects of Mbn manufacture, handling, and regulation remain unclear. In addition, other natural products that have been proposed to mediate copper uptake as well as metallophores that have biologically relevant roles involving copper binding, but not copper uptake, are discussed.

Structural basis of colibactin activation by the ClbP peptidase.

Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-D-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.

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.

MbnH is a diheme MauG-like protein associated with microbial copper homeostasis.

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.

A Peroxodiiron(III/III) Intermediate Mediating Both N-Hydroxylation Steps in Biosynthesis of the N-Nitrosourea Pharmacophore of Streptozotocin by the Multi-domain Metalloenzyme SznF.

The alkylating warhead of the pancreatic cancer drug streptozotocin (SZN) contains an N-nitrosourea moiety constructed from Nω-methyl-l-arginine (l-NMA) by the multi-domain metalloenzyme SznF. The enzyme's central heme-oxygenase-like (HO-like) domain sequentially hydroxylates Nδ and Nω' of l-NMA. Its C-terminal cupin domain then rearranges the triply modified arginine to Nδ-hydroxy-Nω-methyl-Nω-nitroso-l-citrulline, the proposed donor of the functional pharmacophore. Here we show that the HO-like domain of SznF can bind Fe(II) and use it to capture O2, forming a peroxo-Fe2(III/III) intermediate. This intermediate has absorption- and Mössbauer-spectroscopic features similar to those of complexes previously trapped in ferritin-like diiron oxidases and oxygenases (FDOs) and, more recently, the HO-like fatty acid oxidase UndA. The SznF peroxo-Fe2(III/III) complex is an intermediate in both hydroxylation steps, as shown by the concentration-dependent acceleration of its decay upon exposure to either l-NMA or Nδ-hydroxy-Nω-methyl-l-Arg (l-HMA). The Fe2(III/III) cluster produced upon decay of the intermediate has a small Mössbauer quadrupole splitting parameter, implying that, unlike the corresponding product states of many FDOs, it lacks an oxo-bridge. The subsequent decomposition of the product cluster to one or more paramagnetic Fe(III) species over several hours explains why SznF was previously purified and crystallographically characterized without its cofactor. Programmed instability of the oxidized form of the cofactor appears to be a unifying characteristic of the emerging superfamily of HO-like diiron oxidases and oxygenases (HDOs).

Methanobactins: Maintaining copper homeostasis in methanotrophs and beyond.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified natural products that bind copper with high affinity and specificity. Originally identified in methanotrophic bacteria, which have a high need for copper, operons encoding these compounds have also been found in many non-methanotrophic bacteria. The proteins responsible for Mbn biosynthesis include several novel enzymes. Mbn transport involves export through a multidrug efflux pump and re-internalization via a TonB-dependent transporter. Release of copper from Mbn and the molecular basis for copper regulation of Mbn production remain to be elucidated. Future work is likely to result in the identification of new enzymatic chemistry, opportunities for bioengineering and drug targeting of copper metabolism, and an expanded understanding of microbial metal homeostasis.

Methanobactin transport machinery.

Methanotrophic bacteria use methane, a potent greenhouse gas, as their primary source of carbon and energy. The first step in methane metabolism is its oxidation to methanol. In almost all methanotrophs, this chemically challenging reaction is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent integral membrane enzyme. Methanotrophs acquire copper (Cu) for pMMO by secreting a small ribosomally produced, posttranslationally modified natural product called methanobactin (Mbn). Mbn chelates Cu with high affinity, and the Cu-loaded form (CuMbn) is reinternalized into the cell via an active transport process. Bioinformatic and gene regulation studies suggest that two proteins might play a role in CuMbn handling: the TonB-dependent transporter MbnT and the periplasmic binding protein MbnE. Disruption of the gene that encodes MbnT abolishes CuMbn uptake, as reported previously, and expression of MbnT in Escherichia coli confers the ability to take up CuMbn. Biophysical studies of MbnT and MbnE reveal specific interactions with CuMbn, and a crystal structure of apo MbnE is consistent with MbnE's proposed role as a periplasmic CuMbn transporter. Notably, MbnT and MbnE exhibit different levels of discrimination between cognate and noncognate CuMbns. These findings provide evidence for CuMbn-protein interactions and begin to elucidate the molecular mechanisms of its recognition and transport.

Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies.

The CopC Family: Structural and Bioinformatic Insights into a Diverse Group of Periplasmic Copper Binding Proteins.

The CopC proteins are periplasmic copper binding proteins believed to play a role in bacterial copper homeostasis. Previous studies have focused on CopCs that are part of seven-protein Cop or Pco systems involved in copper resistance. These canonical CopCs contain distinct Cu(I) and Cu(II) binding sites. Mounting evidence suggests that CopCs are more widely distributed, often present only with the CopD inner membrane protein, frequently as a fusion protein, and that the CopC and CopD proteins together function in the uptake of copper to the cytoplasm. In the methanotroph Methylosinus trichosporium OB3b, genes encoding a CopCD pair are located adjacent to the particulate methane monooxygenase (pMMO) operon. The CopC from this organism (Mst-CopC) was expressed, purified, and structurally characterized. The 1.46 Å resolution crystal structure of Mst-CopC reveals a single Cu(II) binding site with coordination somewhat different from that in canonical CopCs, and the absence of a Cu(I) binding site. Extensive bioinformatic analyses indicate that the majority of CopCs in fact contain only a Cu(II) site, with just 10% of sequences corresponding to the canonical two-site CopC. Accordingly, a new classification scheme for CopCs was developed, and detailed analyses of the sequences and their genomic neighborhoods reveal new proteins potentially involved in copper homeostasis, providing a framework for expanded models of CopCD function.

Characterization of Methanobactin from Methylosinus sp. LW4.

Methanobactins (Mbns) are a growing family of ribosomally produced, post-translationally modified natural products. Characteristic nitrogen-containing heterocycles and neighboring thioamides allow these compounds to bind copper with high affinity. Genome mining has enabled the identification of Mbn operons in bacterial genomes and the prediction of diverse Mbn structures from operon content and precursor peptide sequence. Here we report the characterization of Mbn from Methylosinus (Ms.) species (sp.) LW4. The peptide backbone is distinct from all previously characterized Mbns, and the post-translational modifications correspond precisely to those predicted on the basis of the Ms. sp. LW4 Mbn operon. Thus, prediction based on genome analysis combined with isolation and structural characterization represents a phylogenetic approach to finding diverse Mbns and elucidating their biosynthetic pathways.

Genome mining for methanobactins.

BACKGROUND: Methanobactins (Mbns) are a family of copper-binding natural products involved in copper uptake by methanotrophic bacteria. The few Mbns that have been structurally characterized feature copper coordination by two nitrogen-containing heterocycles next to thioamide groups embedded in a peptidic backbone of varying composition. Mbns are proposed to derive from post-translational modification of ribosomally synthesized peptides, but only a few genes encoding potential precursor peptides have been identified. Moreover, the relevance of neighboring genes in these genomes has been unclear. RESULTS: The potential for Mbn production in a wider range of bacterial species was assessed by mining microbial genomes. Operons encoding Mbn-like precursor peptides, MbnAs, were identified in 16 new species, including both methanotrophs and, surprisingly, non-methanotrophs. Along with MbnA, the core of the operon is formed by two putative biosynthetic genes denoted MbnB and MbnC. The species can be divided into five groups on the basis of their MbnA and MbnB sequences and their operon compositions. Additional biosynthetic proteins, including aminotransferases, sulfotransferases and flavin adenine dinucleotide (FAD)-dependent oxidoreductases were also identified in some families. Beyond biosynthetic machinery, a conserved set of transporters was identified, including MATE multidrug exporters and TonB-dependent transporters. Additional proteins of interest include a di-heme cytochrome c peroxidase and a partner protein, the roles of which remain a mystery. CONCLUSIONS: This study indicates that Mbn-like compounds may be more widespread than previously thought, but are not present in all methanotrophs. This distribution of species suggests a broader role in metal homeostasis. These data provide a link between precursor peptide sequence and Mbn structure, facilitating predictions of new Mbn structures and supporting a post-translational modification biosynthetic pathway. In addition, testable models for Mbn transport and for methanotrophic copper regulation have emerged. Given the unusual modifications observed in Mbns characterized thus far, understanding the roles of the putative biosynthetic proteins is likely to reveal novel pathways and chemistry.

Structure and function of prodrug-activating peptidases.

Bacteria protect themselves from the toxicity of antimicrobial metabolites they produce through several strategies. In one resistance mechanism, bacteria assemble a non-toxic precursor on an N-acyl-d-asparagine prodrug motif in the cytoplasm, then export it to the periplasm where a dedicated d-amino peptidase hydrolyzes the prodrug motif. These prodrug-activating peptidases contain an N-terminal periplasmic S12 hydrolase domain and C-terminal transmembrane domains (TMDs) of varying lengths: type I peptidases contain three transmembrane helices, and type II peptidases have an additional C-terminal ABC half-transporter. We review studies which have addressed the role of the TMD in function, the substrate specificity, and the biological assembly of ClbP, the type I peptidase that activates colibactin. We use modeling and sequence analyses to extend those insights to other prodrug-activating peptidases and ClbP-like proteins which are not part of prodrug resistance gene clusters. These ClbP-like proteins may play roles in the biosynthesis or degradation of other natural products, including antibiotics, may adopt different TMD folds, and have different substrate specificity compared to prodrug-activating homologs. Finally, we review the data supporting the long-standing hypothesis that ClbP interacts with transporters in the cell and that this association is important for the export of other natural products. Future investigations of this hypothesis as well as of the structure and function of type II peptidases will provide a complete account of the role of prodrug-activating peptidases in the activation and secretion of bacterial toxins.

Dual pathways for copper uptake by methanotrophic bacteria.

Methanobactin (Mb), a 1217-Da copper chelator produced by the methanotroph Methylosinus trichosporium OB3b, is hypothesized to mediate copper acquisition from the environment, particularly from insoluble copper mineral sources. Although indirect evidence suggests that Mb provides copper for the regulation and activity of methane monooxygenase enzymes, experimental data for direct uptake of copper loaded Mb (Cu-Mb) are lacking. Uptake of intact Cu-Mb by M. trichosporium OB3b was demonstrated by isotopic and fluorescent labeling experiments. Confocal microscopy data indicate that Cu-Mb is localized in the cytoplasm. Both Cu-Mb and unchelated Cu are taken up by M. trichosporium OB3b, but by different mechanisms. Uptake of unchelated Cu is inhibited by spermine, suggesting a porin-dependent passive transport process. By contrast, uptake of Cu-Mb is inhibited by the uncoupling agents carbonyl cyanide m-chlorophenylhydrazone and methylamine, but not by spermine, consistent with an active transport process. Cu-Mb from M. trichosporium OB3b can also be internalized by other strains of methanotroph, but not by Escherichia coli, suggesting that Cu-Mb uptake is specific to methanotrophic bacteria. These findings are consistent with a key role for Cu-Mb in copper acquisition by methanotrophs and have important implications for further investigation of the copper uptake machinery.

Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation.

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.

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.

A Genomic Toolkit for the Mechanistic Dissection of Intractable Human Gut Bacteria.

Despite the remarkable microbial diversity found within humans, our ability to link genes to phenotypes is based upon a handful of model microorganisms. We report a comparative genomics platform for Eggerthella lenta and other Coriobacteriia, a neglected taxon broadly relevant to human health and disease. We uncover extensive genetic and metabolic diversity and validate a tool for mapping phenotypes to genes and sequence variants. We also present a tool for the quantification of strains from metagenomic sequencing data, enabling the identification of genes that predict bacterial fitness. Competitive growth is reproducible under laboratory conditions and attributable to intrinsic growth rates and resource utilization. Unique signatures of in vivo competition in gnotobiotic mice include an adhesin enriched in poor colonizers. Together, these computational and experimental resources represent a strong foundation for the continued mechanistic dissection of the Coriobacteriia and a template that can be applied to study other genetically intractable taxa.

Native top-down mass spectrometry provides insights into the copper centers of membrane-bound methane monooxygenase.

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.

Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria.

Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide. Here we show that PmoD, a protein encoded within many pmo operons that is homologous to the AmoD proteins encoded within AOB amo operons, forms a copper center that exhibits the features of a well-defined CuA site using a previously unobserved ligand set derived from a cupredoxin homodimer. PmoD is critical for copper-dependent growth on methane, and genetic analyses strongly support a role directly related to pMMO and AMO. These findings identify a copper-binding protein that may represent a missing link in the function of enzymes critical to the global carbon and nitrogen cycles.

The biosynthesis of methanobactin.

Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.

Methanobactins: from genome to function.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptide (RiPP) natural products that bind copper with high affinity using nitrogen-containing heterocycles and thioamide groups. In some methanotrophic bacteria, Mbns are secreted under conditions of copper starvation and then re-internalized as a copper source for the enzyme particulate methane monooxygenase (pMMO). Genome mining studies have led to the identification and classification of operons encoding the Mbn precursor peptide (MbnA) as well as a number of putative transport, regulatory, and biosynthetic proteins. These Mbn operons are present in non-methanotrophic bacteria as well, suggesting a broader role in and perhaps beyond copper acquisition. Genetic and biochemical studies indicate that specific operon-encoded proteins are involved in Mbn transport and provide insight into copper-responsive gene regulation in methanotrophs. Mbn biosynthesis is not yet understood, but combined analysis of Mbn structures, MbnA sequences, and operon content represents a powerful approach to elucidating the roles of specific biosynthetic enzymes. Future work will likely lead to the discovery of unique pathways for natural product biosynthesis and new mechanisms of microbial metal homeostasis.