A single shell protein plays a major role in choline transport across the shell of the choline utilization microcompartment of Escherichia coli 536.

Bacterial microcompartments (MCPs) are widespread protein-based organelles that play important roles in the global carbon cycle and in the physiology of diverse bacteria, including a number of pathogens. MCPs consist of metabolic enzymes encapsulated within a protein shell. The main roles of MCPs are to concentrate enzymes together with their substrates (to increase reaction rates) and to sequester harmful metabolic intermediates. Prior studies indicate that MCPs have a selectively permeable protein shell, but the mechanisms that allow selective transport across the shell are not fully understood. Here we examine transport across the shell of the choline utilization (Cut) MCP of Escherichia coli 536, which has not been studied before. The shell of the Cut MCP is unusual in consisting of one pentameric and four hexameric bacterial microcompartment (BMC) domain proteins. It lacks trimeric shell proteins, which are thought to be required for the transport of larger substrates and enzymatic cofactors. In addition, its four hexameric BMC domain proteins are very similar in amino acid sequence. This raises questions about how the Cut MCP mediates the selective transport of the substrate, products and cofactors of choline metabolism. In this report, site-directed mutagenesis is used to modify the central pores (the main transport channels) of all four Cut BMC hexamers to assess their transport roles. Our findings indicate that a single shell protein, CmcB, plays the major role in choline transport across the shell of the Cut MCP and that the electrostatic properties of the CmcB pore also impact choline transport. The implications of these findings with regard to the higher-order structure of MCPs are discussed.

Crystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2.

Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Šresolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.

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.

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.

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.

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.

γ-Aminobutyric Acid Type A Receptor Modulation by Etomidate Analogs.

BACKGROUND: Etomidate is a highly potent anesthetic agent that is believed to produce hypnosis by enhancing γ-aminobutyric acid type A (GABAA) receptor function. The authors characterized the GABAA receptor and hypnotic potencies of etomidate analogs. The authors then used computational techniques to build statistical and graphical models that relate the potencies of these etomidate analogs to their structures to identify the specific molecular determinants of potency. METHODS: GABAA receptor potencies were defined with voltage clamp electrophysiology using α1ß3γ2 receptors harboring a channel mutation (α1[L264T]) that enhances anesthetic sensitivity (n = 36 to 60 measurements per concentration-response curve). The hypnotic potencies of etomidate analogs were defined using a loss of righting reflexes assay in Sprague Dawley rats (n = 9 to 21 measurements per dose-response curve). Three-dimensional quantitative structure-activity relationships were determined in silico using comparative molecular field analysis. RESULTS: The GABAA receptor and hypnotic potencies of etomidate and the etomidate analogs ranged by 91- and 53-fold, respectively. These potency measurements were significantly correlated (r = 0.72), but neither measurement correlated with drug hydrophobicity (r = 0.019 and 0.005, respectively). Statistically significant and predictive comparative molecular field analysis models were generated, and a pharmacophore model was built that revealed both the structural elements in etomidate analogs associated with high potency and the interactions that these elements make with the etomidate-binding site. CONCLUSIONS: There are multiple specific structural elements in etomidate and etomidate analogs that mediate GABAA receptor modulation. Modifying any one element can alter receptor potency by an order of magnitude or more.