Mechanism for the Hydrolysis of a Sulfur-Sulfur Bond Based on the Crystal Structure of the Thiosulfohydrolase SoxB

SoxB is an essential component of the bacterial Sox sulfur oxidation pathway. SoxB contains a di-manganese(II) site and is proposed to catalyze the release of sulfate from a protein-bound cysteine S-thiosulfonate. A direct assay for SoxB activity is described. The structure of recombinant Thermus thermophilus SoxB was determined by x-ray crystallography to a resolution of 1.5 Å. Structures were also determined for SoxB in complex with the substrate analogue thiosulfate and in complex with the product sulfate. A mechanistic model for SoxB is proposed based on these structures.The oxidation of reduced inorganic sulfur species by sulfur bacteria is an important component of the biogeochemical sulfur cycle and has practical applications in biomining, agriculture, biocorrosion, fuel desulfuration, and waste treatment (1, 2). Sulfur bacteria use the electrons liberated in sulfur oxidation reactions as the reductant for carbon dioxide fixation and/or as donors to respiratory electron transport chains.The Sox (sulfur oxidizing) system is one of the most widely distributed sulfur oxidation pathways and is found in both photosynthetic and nonphotosynthetic sulfur-oxidizing eubacteria (3). Substrates of the Sox system are reported to include thiosulfate, sulfide, elemental sulfur, sulfite, and tetrathionate (4–6). The Sox pathway has been best characterized in the α-Proteobacterium Paracoccus pantotrophus. In this bacterium thiosulfate is oxidized to sulfate by the four periplasmic protein complexes SoxYZ, SoxAX, SoxB, and SoxCD (3, 7, 8). Intermediates in the pathway are covalently bound to a cysteine residue located in a conserved Gly-Gly-Cys-Gly-Gly sequence at the C terminus of the SoxY protein (9). This C-terminal peptide acts as a swinging arm enabling the cysteine and its bound adducts to enter the active sites of the other pathway components (10). In the current pathway model the heme protein SoxAX (11) oxidatively conjugates thiosulfate to the SoxY swinging arm to form a cysteine S-thiosulfonate, which is then degraded by a combination of SoxB and SoxCD. The electrons produced in the two oxidative steps are fed into the electron transfer chain via a small c-type cytochrome. Many bacteria with a Sox system lack the SoxCD complex found in P. pantotrophus and are instead thought to feed the sulfane group of thiosulfate into other sulfur oxidation pathways (12–14).The reaction assigned to SoxB in the Sox pathway model is the hydrolysis of a sulfur-sulfur bond. This is an unusual enzymatic reaction that has only otherwise been suggested for enzymes designated as trithionate or tetrathionate hydrolases (15–18). The thiosulfohydrolase activity proposed for SoxB has yet to be directly demonstrated. It is, instead, inferred from two key observations. First, in vitro pathway reconstitution experiments show that SoxB catalyzes a nonoxidative reaction (7). Second, SoxB has sequence similarity to the 5′-nucleotidase family of enzymes (19). Because 5′-nucleotidases catalyze the hydrolytic cleavage of phosphate groups from nucleotides, this sequence similarity suggests that SoxB also carries out a hydrolytic reaction.Catalytically active SoxB purified from P. pantotrophus or the closely related bacterium Paracoccus versutus contains up to two atoms of manganese but only traces of other metal ions (20, 21). EPR studies suggest that the manganese ions are present in the form of a dinuclear Mn(II) cluster with bis(μ-hydroxo) (μ-carboxylato) bridging ligands (20, 22).In phylogenetic and environmental studies the presence of a soxB gene has been used as a marker for the presence of the Sox pathway and as an indicator of the ability of the organism to oxidize thiosulfate (23, 24).Here we report experiments aimed at establishing a direct assay of SoxB activity. We have used x-ray crystallography to determine the structure of recombinant SoxB from the thermophilic bacterium Thermus thermophilus. This is the first structure of an enzyme catalyzing the hydrolysis of a sulfur-sulfur bond. We have also obtained structures of T. thermophilus SoxB in complex with mechanistically relevant ligands. Based on these structures, we propose a model for the SoxB mechanism.     

Electrochemical Measurement of Electron Transfer Kinetics by Shewanella oneidensis MR-1

Shewanella oneidensis strain MR-1 can respire using carbon electrodes and metal oxyhydroxides as electron acceptors, requiring mechanisms for transferring electrons from the cell interior to surfaces located beyond the cell. Although purified outer membrane cytochromes will reduce both electrodes and metals, S. oneidensis also secretes flavins, which accelerate electron transfer to metals and electrodes. We developed techniques for detecting direct electron transfer by intact cells, using turnover and single turnover voltammetry. Metabolically active cells attached to graphite electrodes produced thin (submonolayer) films that demonstrated both catalytic and reversible electron transfer in the presence and absence of flavins. In the absence of soluble flavins, electron transfer occurred in a broad potential window centered at ∼0 V (versus standard hydrogen electrode), and was altered in single (ΔomcA, ΔmtrC) and double deletion (ΔomcA/ΔmtrC) mutants of outer membrane cytochromes. The addition of soluble flavins at physiological concentrations significantly accelerated electron transfer and allowed catalytic electron transfer to occur at lower applied potentials (−0.2 V). Scan rate analysis indicated that rate constants for direct electron transfer were slower than those reported for pure cytochromes (∼1 s⁻¹). These observations indicated that anodic current in the higher (>0 V) window is due to activation of a direct transfer mechanism, whereas electron transfer at lower potentials is enabled by flavins. The electrochemical dissection of these activities in living cells into two systems with characteristic midpoint potentials and kinetic behaviors explains prior observations and demonstrates the complementary nature of S. oneidensis electron transfer strategies.Respiratory electron flow typically occurs at the inner membrane, where oxidation and reduction can be easily linked to intracellular electron carriers and used to generate a membrane potential. However, when the electron acceptor or donor is insoluble, bacteria must possess a mechanism for transferring electrons beyond their inner membrane (1). This is especially true for Proteobcteria, which have an outer membrane that further insulates cytoplasmic and inner membrane processes from insoluble substrates. Metal oxides (such as Fe(III) and Mn(IV) oxyhydroxides) are well recognized naturally occurring electron acceptors that demand such an electron transfer strategy (2–4).Shewanella oneidensis MR-1, a metabolically versatile member of the gammaproteobacteria (5), is capable of reducing insoluble metals, and this phenotype has been linked to a collection of interacting multiheme cytochromes spanning the inner membrane, periplasmic space, and outer membrane (6–12). There is also evidence that some of these cytochromes decorate the surface of pili-like structures extending from the cell surface (13, 14). Regardless of the ultimate location of the cytochromes, in all models of electron transfer, electrons must hop from these proteins to a solid surface or be transferred to a soluble mediator that can diffuse to a final destination (15, 16). Although chelation of a metal oxide is a third option (17, 18), the fact that Shewanella is able to transfer electrons to solid graphite electrodes (19–23) underscores the need for a direct or diffusion-based electron transfer mechanism to link cellular proteins and surfaces.Recent work has shown that Shewanella species secrete soluble flavins (FMN and riboflavin) that facilitate electron transfer to both metals and electrodes (23, 24). For example, removal of accumulated soluble flavins decreases the rate of electron transfer by Shewanella biofilms to electrodes over 80%. Consistent with this observation, kinetic measurements with pure MtrC and OmcA (25) showed that direct reduction of solid metal oxides by these cytochromes was too slow to explain physiological rates of electron transfer, whereas turnover rates of these enzymes with soluble flavins were orders of magnitude larger. These studies suggest that the kinetics of electron transfer from cytochromes on the outer surface of Shewanella to electrodes will be significantly altered in the absence of diffusible mediators (9–11, 26–34).Voltammetry has proven a useful technique for the analysis of electron transfer rates and pathways using purified proteins (35–39) and has recently been extended to the study of intact bacteria (23, 40–42). In slow scan rate cyclic voltammetry, the rate of electron transfer from respiring Shewanella biofilms to electrodes rises sharply at the E°′ of riboflavin and FMN (−0.2 V versus SHE)² (23). Such measurements relating thermodynamic driving force to turnover kinetics would be difficult with whole cell:Fe(III) oxide incubations, which do not allow fine control over the electron acceptor redox potential or real time recording of electron transfer rates. In addition, voltammetry provides tools to observe electron movement under single-turnover conditions (in the absence of electron donor), allowing reversible oxidation and reduction of proteins accessible to the electrode and study of kinetic behavior (43, 44).In this work, techniques of turnover (sustained electron transfer from cells to electrode in the presence of electron donor) and single turnover (reversible oxidation and reduction in the absence of electron donor) voltammetry were harnessed to investigate the role of outer membrane proteins in electron transfer from Shewanella to electrodes. In all of these studies, intact metabolically active cells were used, along with electrode surfaces known to act as acceptors for Shewanella. The results in the absence of soluble mediators provide evidence that electron transfer between MtrC and OmcA and surfaces requires a higher potential compared with when flavins are present to shuttle electrons to the surface. Mutant analysis also demonstrates that cells possessing different outer membrane cytochromes have differing abilities for direct and mediator-enabled electron transfer.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

A Functional P2X7 Splice Variant with an Alternative Transmembrane Domain 1 Escapes Gene Inactivation in P2X7 Knock-out Mice

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-d-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7^−/− mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7^−/− strains has important implications for our understanding of the role of this receptor in health and disease.P2X receptors (P2XRs)³ are ATP-gated cation channels. They consist of three subunits (1, 2) each containing two transmembrane domains (TMDs) linked by an extracellular ligand-binding domain (3). The P2X7 receptor is distinguished from other P2X receptors by its long intracellular C terminus, a low ATP sensitivity (EC₅₀: 100 μm to 1 mm), and its ability to induce “cell permeabilization,” meaning that upon prolonged ATP application the opening of a permeation pathway for large molecules is induced. This process eventually leads to apoptosis, requires the C terminus (3–6), and may be mediated by interaction with pannexin hemichannels (7). In addition, “pore dilation,” which allows the passage of the large cation NMDG, is observed if extracellular sodium is replaced by NMDG (8), a property also displayed by the P2X2 (9) and P2X4 (10) receptors. This pore dilation is assumed to represent an intrinsic property of these P2X receptors (11, 12), although it can be influenced by interaction with intracellular proteins (13). However, both processes are still poorly understood.P2X7 receptors are found on cells of the hematopoietic lineage, in epithelia, and endothelia. Several studies report its expression and/or function in neurons, although its presence here is under debate (14, 15). So far, nine splice variants (P2X7(b) through P2X7(j)) have been described, only one of which was shown to be, at least partially, functional (16, 17). In addition, numerous single nucleotide polymorphisms have been identified in the human P2X7 receptor. Some of these have been found to cause either gain or loss of function and have been associated with chronic lymphocytic leukemia, bone fracture risk, and impaired immune functions (18–20). Recent genetic studies indicate an association between the Gln-460 → Arg polymorphism and familial depressive disorders (21).Two P2X7-deficient mouse lines have been described. In the mouse line generated by Glaxo, the P2rx7 gene was knocked out by insertion of a lacZ transgene into exon 1 (22). In the mouse line generated by Pfizer (23) a neomycin cassette was inserted into exon 13, replacing a region that encodes Cys-506–Pro-532 of the intracellular C terminus of the receptor. The Pfizer P2X7 KO mice demonstrated the involvement of this receptor in bone formation (24), cytokine production, and inflammation (23, 25) while the Glaxo^−/− mice established its role in inflammatory and neuropathic pain (26). All these findings and multiple subsequent studies based on these mouse models defined the P2X7R as a promising target for the development of innovative therapeutic strategies, and selective P2X7 inhibitors are already in clinical trials for the treatment of rheumatoid arthritis (27).Here, we describe a novel P2X7 isoform with an alternative N terminus and TMD 1. Compared with the originally identified P2X7(a) variant, it has increased agonist sensitivity and a higher propensity to form NMDG-permeable pores and permit dye uptake. Due to a novel alternative exon 1 and translation start, this splice variant escapes inactivation in the Glaxo P2X7^−/− mice and thus could account for possible inconsistencies between results obtained with different P2X7^−/− mouse lines (28).     

Copper-Binding Compounds from Methylosinus trichosporium OB3b

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMO^C) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701–3708, 1992), of Methylosinus trichosporium OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L₁ and CBC-L₂, respectively. The amino acid sequence of CBC-L₁ is S?MYPGS?M, and that of CBC-L₂ is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L₁ showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g_⊥ = 2.087, g_∥ = 2.42 G, |A_∥| = 128 G). The EPR spectrum of CBC-L₂ was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu²⁺ center (g_⊥ = 2.073, g_∥ = 2.324 G, |A_∥| = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g_⊥ = 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMO^C mutants PP319 and PP359 regardless of the copper concentration in the culture medium.In methanotrophs, the relationship between the concentration of copper and expression of the two different methane monooxygenases (MMOs) is well characterized (8, 11, 45, 49, 50). Under low copper-to-biomass ratios, methane oxidation activity is observed in the soluble fraction, and the enzyme is referred to as the soluble methane monooxygenase (sMMO). At higher copper-to-biomass ratios, methane oxidation activity is observed in the membrane fraction, and the enzyme is referred to as the membrane-associated or particulate methane monooxygenase (pMMO). The polypeptides and structural genes for both enzymes have been characterized (4, 18–22, 24, 25, 32, 34–40, 43–45, 47–49, 51, 62, 63). In addition to expression of the two MMOs, four other physiological traits have been identified in cells expressing the pMMO that are affected by the copper concentration in the culture medium. First, the concentration of copper in the culture media is directly related to pMMO activity in cell-free fractions, although the levels of expression of pMMO polypeptides vary in different methanotrophs (1, 8, 30, 36, 50, 63). For example, the expression levels of the three pMMO polypeptides in Methylococcus capsulatus Bath remained constant with varying copper concentrations (8, 36), whereas in Methylomicrobium albus BG8, the expression level of the putative pMMO polypeptides increased with increased copper in the culture medium (8). Second, the concentrations of membrane-associated copper and iron show a proportional increase as the copper concentration in the culture medium is increased (36, 63). Third, the formation and level of intracytoplasmic membranes in cells cultured in copper-supplemented media are dependent on the copper concentration in the culture media (8, 11, 40, 48). Lastly, the K_s for methane oxidation by pMMO is altered by the copper concentration in the culture media (33a).Berson and Lidstrom (1) have recently noted that in spite of the central role of copper in the physiology of methanotrophs, the mechanism(s) of copper acquisition remains vague. Although true, a few studies have suggested the existence of a specific copper acquisition system in M. capsulatus Bath and M. trichosporium OB3b. The first indication of a specific copper uptake system was provided from phenotypic characterization of the constitutive sMMO mutants (sMMO^C) isolated by Phelps et al. (42). Fitch et al. (17) found that in M. trichosporium OB3b, these sMMO^C mutants were defective in copper uptake and showed preliminary evidence for an extracellular copper-complexing agent. Working with the same mutants, Téllez et al. partially purified this copper-complexing agent and determined that it was a small molecule with a molecular mass of approximately 500 Da with an association constant with copper of 1.4 × 10¹⁶ M⁻¹ (55). Other evidence for a specific copper uptake system was provided by the copper-binding cofactor (CBC) from M. capsulatus Bath (63). During the isolation of the pMMO from M. capsulatus Bath, CBC was identified in association with the purified enzyme, in the washed membrane fraction, and in the extracellular fraction. The CBC was determined to be a small polypeptide with a molecular mass of 1,232 Da. In M. capsulatus Bath, the cellular location of the CBC varied depending on the copper concentration in the culture medium and on the expression of the pMMO.This paper ties together and extends these observations on specific copper acquisition systems in M. trichosporium OB3b and M. capsulatus Bath. Here we describe the initial isolation and characterization of two copper-complexing agents, called CBC-L₁ and CBC-L₂, from the M. trichosporium OB3b wild type and sMMO^C mutant PP319. CBC-L₁ from M. trichosporium OB3b was identical to the CBC previously identified during the isolation of the pMMO from M. capsulatus Bath. This paper is also the first report of a second CBC, CBC-L₂, which may have been present as a contaminant in previous CBC preparations from M. capsulatus Bath. One or both of the CBCs appear to be the same copper-complexing agent partially purified by Téllez et al. (55). Lastly, this report describes the effect of the copper concentration in the culture medium on copper uptake, the expression of both MMOs, and extracellular concentration of the CBC in wild-type and sMMO^C mutant strains of M. trichosporium OB3b.     

Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO₂ reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.Phosphorus (P) is an important nutrient for all living organisms. The predominant forms of phosphorus in biological systems are inorganic phosphate and its organic esters and acid anhydrides in which P is at its highest oxidation state (+V). The P requirements of living cells can be fulfilled with phosphate in various forms, including reduced organic and inorganic phosphorus compounds (23). Several aerobic bacteria were shown to be able to oxidize hypophosphite (+I) and phosphite (+III) to phosphate (+V) and to incorporate the last into their biomass (5, 15-17, 31, 34). Phosphite can also be oxidized under anaerobic conditions, as shown for an anaerobic Bacillus strain (7) and for Pseudomonas stutzeri which can use phosphite under denitrifying conditions (17, 21). The only bacterium known to oxidize phosphite as the sole source of electrons in lithoautotrophic energy metabolism is Desulfotignum phosphitoxidans (24, 25).Three different metabolic pathways for the use of phosphite as a single P source have been characterized so far. Two of them were discovered and characterized with Escherichia coli and one with Pseudomonas stutzeri. The first pathway in E. coli is mediated by the enzyme carbon phosphorus lyase (C-P lyase), and the second one by the alkaline phosphatase encoded by phoA (16, 34). This alkaline phosphatase not only hydrolyzes phosphate esters but also hydrolyzes phosphite to phosphate and molecular hydrogen (32). This is a particular property only of the E. coli alkaline phosphatase and is not observed with alkaline phosphatases of other bacteria. The third pathway is encoded by the ptxABCDE gene cluster in P. stutzeri (17). In this system, phosphite is transported into the cell by a binding protein-dependent phosphite transporter at the expense of ATP (PtxABC). Phosphite is oxidized by a phosphite:NAD⁺ oxidoreductase (encoded by ptxD), a new member of the 2-hydroxy acid dehydrogenases (8). The ptx operon of P. stutzeri is regulated in response to phosphate starvation by the two-component regulatory system phoBR (28, 29). Furthermore, in Alcaligenes faecalis WM2072, another gene cluster involved in hypophosphite and phosphite uptake and oxidation was characterized: the htxABCD-ptxDE locus (31). The htxABCD-ptxDE genes and their products in A. faecalis WM 2072 have high nucleotide and amino acid sequence identities with those found in the htx and ptx operons in P. stutzeri WM88, which are required for the oxidation of hypophosphite and phosphite, respectively. This unique genetic arrangement of hypophosphite- and phosphite-oxidizing genes in A. faecalis WM2072 suggests a horizontal gene transfer and an ancient evolution of phosphite oxidation.The diversity of pathways used for assimilatory phosphite oxidation and the fact that D. phosphitoxidans is so far the only bacterium known to use phosphite as an electron source caused us to investigate the phosphite uptake and oxidation gene cluster of this bacterium. The aims of our study were (i) to establish enzymatic assays for measurement of phosphite oxidation activity in cell extracts, (ii) to identify the genes involved in phosphite uptake and oxidation, and (iii) to characterize these genes physiologically.     

Evidence of Highly Conserved β-Crystallin Disulfidome that Can be Mimicked by In Vitro Oxidation in Age-related Human Cataract and Glutathione Depleted Mouse Lens*

Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract. To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- versus intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ∼4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H₂O₂, whereas γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33, and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130, and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in γC; Cys42, Cys33 in γD, and Cys83, Cys115, and Cys130 in γS. In conclusion, the β-crystallin disulfidome is highly conserved in age-related nuclear cataract and LEGSKO mouse, and reproducible by in vitro oxidation, whereas some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age-related nuclear cataract.Aging lens crystallins accumulate post-synthetic modifications that can be broadly classified into three categories, namely (1) protein backbone changes, such as racemization and truncation (1–3), (2) conversion of one amino acid into another, such as deamidation of asparagine into aspartate or deguanidination of arginine into ornithine, deamination of lysine into allysine and 2-aminoadipic acid (4–6), and (3) amino acid residue damage from reactive carbonyls and reactive oxygen species (7,8). Carbonyl damage results from the Maillard Reaction by glucose, methylglyoxal, or oxidation products of ascorbate, tryptophan or lipids which form adducts and crosslinks with nucleophilic group of lysine, arginine and cysteine. Examples include carboxymethyl-lysine, pentosidine, methylglyoxal hydroimidazolones, HNE-cysteine adducts and kynurenine (7,9–12). Oxidative damage results from reactive oxygen species that directly damage amino acid residues, e.g. oxidizing tryptophan into N-formyl kynurenine and kynurenine, methionine into its sulfoxide, and cysteine into cysteine disulfides or cysteic acid (13–15).Because of their relevance to age-related cataract, the impact of each of these modifications on crystallin structure and stability is the subject of intense investigation. Importantly, Benedek proposed that high molecular weight (HMW)¹ crystallin aggregates the size of 50 million daltons are needed in order for lens opacification to be visible(16,17). Crystallin aggregation conceivably occurs by one of several mechanisms that include conformational changes as a consequence amino acid mutations (18) or physical-chemical protein modifications. Of the latter, one mechanism that is dominant in several types of cataract involves oxidation of cysteines into protein disulfides (18) and formation of HMW aggregates that scatter light (19).In order to mimic the oxidative process and formation of protein disulfides linked to low concentrations of glutathione (GSH) in the nucleus of the human lens, we recently created the LEGSKO mouse in which lenticular GSH was lowered by knocking out the γ-glutamyl cysteine ligase subunit Gclc (20). These mice develop full-blown nuclear cataract by about 9 months and represent an important model for the development of drugs that might block or reverse the oxidation of crystallin sulfhydryls and presumably protein aggregation. However, this assumption in part depends on whether the sites of disulfide bond formation are similar in mouse and human age-related cataract. To test this hypothesis we performed the first comparative analysis of the cataract prone LEGSKO mouse and human aging and cataractous lens crystallin disulfidome, and compared the results with the disulfidome from mouse lens homogenate oxidized in vitro with H₂O₂ as a model of crystallin aggregation and opacification.     

Hypochlorous Acid Converts the ��-Glutamyl Group of Glutathione Disulfide to 5-Hydroxybutyrolactam, a Potential Marker for Neutrophil Activation

In healthy cells, glutathione disulfide (GSSG) is rapidly reduced back to glutathione (GSH) by glutathione reductase to maintain redox status. The ratio of GSH/GSSG has been used as an indicator of oxidative stress. However, hypochlorous acid (HOCl) generated by the myeloperoxidase-H₂O₂-Cl⁻ system of neutrophils converts GSH to irreversible oxidation products. Although several such products have been identified, yields of these compounds are very low in biological systems, and they cannot account quantitatively for thiol loss. In the current studies, we use liquid chromatography-mass spectrometry (LC-MS) to demonstrate that HOCl and chloramines oxidize GSSG to two irreversible products in high yield. The products, termed M-45 and M-90, are, respectively, 45 or 90 atomic mass units lighter than GSSG. The reaction pathway involves chloramine and aldehyde intermediates, and converts the γ-glutamyl residues of GSSG to 5-hydroxybutyrolactam. Importantly, M-45 and M-90 were resistant to reduction by glutathione reductase. Moreover, the monohydroxylbutyrolactam M-45 accounted for >90% of the endogenous GSH oxidation products generated by activated neutrophils. Because the reaction pathway involves chlorinating intermediates, hydroxylbutyrolactams are likely to be specific products of HOCl, which is generated only by myeloperoxidase. Therefore, our observations implicate M-45 as a potential biomarker for myeloperoxidase activity in vivo.Glutathione (GSH), a tripeptide synthesized in the cytosol from glutamate, cysteine, and glycine, is the predominant antioxidant in mammalian cells. Its concentration ranges from millimolar inside cells to micromolar in plasma (1, 2). In many cells, GSH accounts for >90% of total nonprotein thiol (3, 4). The free thiol group in GSH is responsible for biological activity. As a nucleophilic scavenger, GSH can directly react with electrophilic substances, such as reactive oxygen/nitrogen species, or be oxidized by GSH peroxidase to glutathione disulfide (GSSG). Therefore, it is essential for maintaining intracellular redox status and defending against oxidative injury. Under normal circumstances, GSSG is rapidly reduced back to GSH by glutathione reductase and NADPH. Thus, most of the GSH remains in the reduced form. Under oxidative stress, however, GSH is converted to GSSG, which potentially accumulates (2, 5). Indeed, the GSH/GSSG ratio has been used to evaluate oxidative stress in biological systems. Alterations of this ratio associate with a variety of diseases, including atherosclerosis, cancer, and human immunodeficiency virus infection (6–10).One important source of oxidative stress in humans is myeloperoxidase (MPO),² a heme protein expressed by neutrophils, monocytes, and certain populations of macrophages (11–13). Activation of these inflammatory white blood cells results in the secretion of MPO, which uses hydrogen peroxide (H₂O₂, produced by NADPH oxidase) and chloride anion to generate hypochlorous acid (HOCl) (14). HOCl rapidly reacts with a wide range of functional groups (15–19). At physiological pH, thiol groups and free amino groups are its main targets, and the initial products are oxidized thiols and chloramines.HOCl generates other products in addition to GSSG when it reacts with GSH. Chesney et al. (20) suggested that it oxidizes GSH to a higher oxidation state than the disulfide form because the molar ratio of HOCl consumed to GSH oxidized was 4:1 instead of 1:1 in Escherichia coli. Winterbourn (21) reported that approximately half of the GSH oxidized by HOCl could not be regenerated. These researchers have identified glutathione sulfonamide (GSA), glutathione thiosulfonate, and dehydroglutathione as irreversible higher oxidation products (22, 23). Their observations suggest that the formation of higher order GSSG oxidation products might account in part for the irreversible loss of GSH induced by HOCl. However, activated neutrophils (the source of MPO and therefore of HOCl) generate only low yields of these higher oxidation products, suggesting that the major products of GSH oxidation by MPO remain to be identified.The disulfide and α-amino groups of GSSG are also potential targets of HOCl (17). Disulfides can be oxidized to sulfonic acid via a sulfenyl chloride intermediate (16). α-Amino groups yield chloramines, which undergo decarboxylation, intramolecular H-abstraction, or other reaction pathways to form various products, such as aldehydes and carboxymethyllysine (16, 24). These reactions may be biologically relevant, because carboxymethyllysine production is impaired in mice deficient in the phagocyte NADPH oxidase (25). These observations suggest that GSSG is a potential scavenger of HOCl. Indeed, GSSG reportedly competes for HOCl with its rate constant expected to be 2 × 10⁵ m⁻¹ s⁻¹ (26, 27). Studies from Bast et al. (28) demonstrated that GSSG protects acetylcholinesterase from oxidative inactivation by HOCl. Nagy and Ashby (29) studied the kinetics and mechanism of GSSG oxidation by HOCl. They proposed that HOCl generates the bis-N-chloro-γ-l-glutamyl derivative of GSSG. These studies suggest that GSSG itself may function as an antioxidant.In the current study, we investigated the reaction of GSSG with HOCl and other oxidants. Using liquid chromatography in concert with mass spectrometry (LC-MS), we identified two groups of novel oxidation products, which we termed M-45 and M-90. We characterized their structures and potential reaction pathways. Our results indicate that HOCl and chloramines oxidize the γ-glutamyl moiety of GSSG to 5-hydroxybutyrolactam in high yield.     

Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1). Chagas'' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2–4).T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5, 6). Inhibitors of cruzain and rhodesain, major cathepsin L-like papain-family cysteine proteases of T. cruzi and T. brucei rhodesiense (7–10) display considerable antitrypanosomal activity (11, 12), and some classes have been shown to cure T. cruzi infection in mouse models (11, 13, 14).In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2)⁶ and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15–17). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17), FP-3 has proved recalcitrant to gene knock-out (16) suggesting a critical function for this enzyme and underscoring its potential as a drug target.Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19–22). Among these small molecules, vinyl sulfones have been shown to be effective inhibitors of a number of papain family-like cysteine proteases (19, 23–27). Vinyl sulfones have many desirable attributes, including selectivity for cysteine proteases over serine proteases, stable inactivation of the target enzyme, and relative inertness in the absence of the protease target active site (25). This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28, 29). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds.     

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a ��-Carotene 15,15��-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The K_m, k_cat, and k_cat/K_m values for β-carotene as substrate were 37 μm, 3.6 min⁻¹, and 97 mm⁻¹ min⁻¹, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₅ seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (3–5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23–28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.     

Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABA_ARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABA_ARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABA_AR (δL2), but not the γGABA_AR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABA_ARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA_A γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA_ARs. The δL2 mutations did not affect GlyR or GABA_AR sensitivity, respectively, to Zn²⁺ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3–5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)² have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6–12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine₃, γ-aminobutyric acid type A (GABA_A), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 15–20) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23–25).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7–12, 18, 19, 26–30). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABA_ARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 34–40).GlyRs and GABA_ARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABA_ARs (e.g. α1β2γ2), the most predominantly expressed GABA_ARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABA_ARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44–51). Sequence alignment of α1GlyR, γGABA_AR, and δGABA_AR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABA_AR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABA_ARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABA_ARs with corresponding non-conserved residues from the δ subunit of GABA_AR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABA_AR subunit (δL2), but not the γGABA_AR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABA_ARs with δL2 also markedly increased ethanol sensitivity of GABA_ARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.

Polychlorinated biphenyls (PCBs) are produced by chlorination of biphenyl, resulting in up to 209 different congeners. Commercial mixtures range from light oily fluids to waxes, and their physical properties make them useful as heat transfer fluids, hydraulic fluids, solvent extenders, plasticizers, flame retardants, organic diluents, and dielectric fluids (1, 21). Approximately 24 million lb are in the North American environment (19). The stability and hydrophobic nature of these compounds make them a persistent environmental hazard.To date, bacterial transformations have been the main focus of PCB degradation research. Aerobic bacteria use a biphenyl-induced dioxygenase enzyme system to attack less-chlorinated congeners (mono- to hexachlorobiphenyls) (1, 5, 7, 8, 22). Although more-chlorinated congeners are recalcitrant to aerobic bacterial degradation, microorganisms in anaerobic river sediments reductively dechlorinate these compounds, mainly removing the meta and para chlorines (1, 6, 10, 33, 34).The degradation of PCBs by white rot fungi has been known since 1985 (11, 18). Many fungi have been tested for their ability to degrade PCBs, including the white rot fungi Coriolus versicolor (18), Coriolopsis polysona (41), Funalia gallica (18), Hirneola nigricans (35), Lentinus edodes (35), Phanerochaete chrysosporium (3, 11, 14, 17, 18, 35, 39, 41–43), Phlebia brevispora (18), Pleurotus ostreatus (35, 43), Poria cinerescens (18), Px strain (possibly Lentinus tigrinus) (35), and Trametes versicolor (41, 43). There have also been studies of PCB metabolism by ectomycorrhizal fungi (17) and other fungi such as Aspergillus flavus (32), Aspergillus niger (15), Aureobasidium pullulans (18), Candida boidinii (35), Candida lipolytica (35), Cunninghamella elegans (16), and Saccharomyces cerevisiae (18, 38). The mechanism of PCB biodegradation has not been definitively determined for any fungi. White rot fungi produce several nonspecific extracellular enzymes which have been the subject of extensive research. These nonspecific peroxidases are normally involved in lignin degradation but can oxidize a wide range of aromatic compounds including polycyclic aromatic hydrocarbons (37). Two peroxidases, lignin peroxidase (LiP) and Mn peroxidase (MnP), are secreted into the environment of the fungus under conditions of nitrogen limitation in P. chrysosporium (23, 25, 27, 29) but are not stress related in fungi such as Bjerkandera adusta or T. versicolor (12, 30).Two approaches have been used to determine the biodegradability of PCBs by fungi: (i) loss of the parent congener analyzed by gas chromatography (GC) (17, 32, 35, 42, 43) and (ii) mineralization experiments in which the ¹⁴C of the universally labeled ¹⁴C parent congener is recovered as ¹⁴CO₂ (11, 14, 18, 39, 41). In the first method, the loss of a peak on a chromatogram makes it difficult to decide whether the PCB is being partly degraded, mineralized, adsorbed to the fungal biomass, or bound to glassware, soil particles, or wood chips. Even when experiments with killed-cell and abiotic controls are performed, the extraction efficiency and standard error can make data difficult to interpret. For example, recoveries can range anywhere from 40 to 100% depending on the congener used and the fungus being investigated (17). On the other hand, recovery of significant amounts of ¹⁴CO₂ from the cultures incubated with a ¹⁴C substrate provides definitive proof of fungal metabolism. There appears to be only one report relating data from these two techniques (18), and in that study, [U-¹⁴C]Aroclor 1254, rather than an individual congener, was used.In this study, we examined the ability of 12 white rot fungal strains to metabolize selected PCB congeners to determine which strains were the most active degraders. Included in this group was P. chrysosporium ATCC 24725, a strain used extensively in PCB studies (3, 14, 18, 35, 39, 41–43). Six PCB congeners were selected to give a range of chlorine substitutions and therefore a range of potential biodegradability which was monitored by GC. One of the chosen congeners was ¹⁴C labeled and used in studies to compare the results from a mineralization method with those from the GC method.