"Green" synthesized versus chemically synthesized zinc oxide nanoparticles: In vivo antihyperglycemic activity and pharmacokinetics.

Zinc is one of the most studied trace elements, commonly used as supplement in diabetes treatment. By its involvement in the synthesis, secretion of insulin, promotion of insulin sensitivity and its multiple enzymatic functions it is known to contribute to reduce hyperglycemia. Researchers have shown that zinc administered under the form of zinc oxide nanoparticles (ZnONPs) is more effective than under its ionic form. Studies evaluating the antihyperglycemic activity of these nanocarriers include both ZnONPs synthesised using plants (i.e. green synthesized) or chemically synthesized. The present work aims to compare green synthesized ZnONPs with the marketed chemically synthesized ones. Green ZnONPs were synthesized using the aqueous extract of the stem bark of the medicinal plant Panda oleosa and zinc nitrate hexahydrate. Both nanocarriers were compared in terms of optical properties, morphology, composition, chemical functions, resistance to oxidation, in vivo antihyperglycemic activity via oral glucose tolerance test (OGTT) and pharmacokinetics in relation to zinc in C57BL/6J mice. A UV absorption peak was observed at 354 nm and 374 nm for the green and marketed ZnONPs, respectively. The shape and hydrodynamic diameters were anisotropic and of 228.8 ± 3.0 nm for the green ZnONPs and spherical and of 225.6 ± 0.9 nm for the marketed ZnONPs. Phenolic compounds accounted for 2.58 ± 0.04% of the green ZnONPs and allowed them to be more stable and unaffected by an oxidizing agent during the experiment, while the marketed chemically synthesized ZnONPs aggregated with or without contact with an oxidizing agent. No significant differences were observed on the amounts of zinc absorbed when comparing green ZnONPs, chemically synthesized ZnONPs and zinc sulfate in a pharmacokinetics study in normoglycemic mice. When evaluating the in vivo hypoglycemic activity of the nanocarriers in obese/diabetic mice, green synthesized ZnONPs displayed a significant hypoglycemic effect compared with the chemically synthesized nanoparticles following an OGTT. Altogether, these data indicate that phytocompounds, as catechin derivatives and polyphenols, attached to the green synthesized ZnONPs' surface, could contribute to their hypoglycemic activity. The comparison thus demonstrated that green synthesized ZnONPs are significantly more efficient than chemically ones at reducing hyperglycemia regardless of their absorption.

Poly-ɛ-caprolactone nanocapsules loaded with copaiba essential oil reduce inflammation and pain in mice.

Diverse drugs have been used for the management of inflammation disorders and pain. However, they present many side effects and stimulate the search for new pharmacotherapeutic alternatives. Plant-derived products such as copaiba essential oil (CO) offer beneficial pharmacological effects. On the other hand, essential oil's low water solubility and physical instability hinder its in vivo application. Thus, poly-ɛ-caprolactone (PCL)-based nanocarriers have been used to increase their stability and efficacy. This work aimed to encapsulate CO in PCL nanocapsules and evaluate their effect on inflammation models and pain. The polymeric nanocapsules loading CO (CO-NC) were prepared by nanoprecipitation technique, characterized, and analyzed for their anti-inflammatory effect in vitro and in vivo. The results showed that CO-NC presented a spherical shape, 229.3 ± 1.5 nm diameter, and a negative zeta potential (approximately -23 mV). CO and CO-NC presented anti-inflammatory and antioxidant effects by LPS-activated macrophages (J774 cells). In addition, CO-NC significantly reduced TNF-α secretion (3-fold) compared to CO. In vivo, pre-treatment with CO or CO-NC (50, 100, 200 mg/kg, intraperitoneal; i.p) reduced the mechanical allodynia, paw edema, and pro-inflammatory cytokines induced by intraplantar (i.pl) injection of carrageenan in mice. Specifically, CO-NC (200 mg/kg; i.p.) reduced the production of TNF-α similar to the control group. Our results support using polymeric nanocapsules for CO delivery in inflammatory conditions.

Mesenchymal stromal cells encapsulated in licensing hydrogels exert delocalized systemic protection against ulcerative colitis via subcutaneous xenotransplantation.

The ability of mesenchymal stromal cells (MSCs) to release a plethora of immunomodulatory factors makes them valuable candidates to overcome inflammatory bowel diseases (IBD). However, this cell therapy approach is still limited by major issues derived from nude MSC-administration, including a rapid loss of their immunomodulatory phenotype that impairs factor secretion, low persistence and impossibility to retrieve the cells in case of adverse effects. Here, we designed a licensing hydrogel system to address these limitations and thus, obtain a continuous delivery of bioactive factors. IFNγ-loaded heparin-coated beads were included in injectable in situ crosslinking alginate hydrogels, providing a 3D microenvironment that ensured continuous inflammatory licensing, cell persistence and implant retrievability. Licensing-hydrogel encapsulated human MSCs (hMSCs) were subcutaneously xenotransplanted in an acute mouse model of ulcerative colitis. Results showed that encapsulated hMSCs exerted a delocalized systemic protection, not presenting significant differences to healthy mice in the disease activity index, colon weight/length ratio and histological score. At day 7, cells were easily retrieved and ex vivo assays showed fully viable hMSCs that retained an immunomodulatory phenotype, as they continued secreting factors including PGE2 and Gal-9. Our data demonstrate the capacity of licensing hydrogel-encapsulated hMSCs to limit the in vivo progression of IBD.

Impact of PEGylation on an antibody-loaded nanoparticle-based drug delivery system for the treatment of inflammatory bowel disease.

Nanoparticle-based oral drug delivery systems have the potential to target inflamed regions in the gastrointestinal tract by specifically accumulating at disrupted colonic epithelium. But, delivery of intact protein drugs at the targeted site is a major challenge due to the harsh gastrointestinal environment and the protective mucus layer. Biocompatible nanoparticles engineered to target the inflamed colonic tissue and efficiently penetrate the mucosal layer can provide a promising approach for orally delivering monoclonal antibodies to treat inflammatory bowel disease. The study aims to develop mucus-penetrating nanoparticles composed of poly(lactic-co-glycolic acid, PLGA) polymers with two different polyethylene glycol (PEG) chain lengths (2 kDa and 5kDa) to encapsulate monoclonal antibody against tumor necrosis factor-α (TNF-α). The impact of different PEG chain lengths on the efficacy of the nanosystems was evaluated in vitro, ex vivo, and in vivo. Both PLGA-PEG2k and PLGA-PEG5k nanoparticles successfully encapsulated the antibody and significantly reduced TNF-α secretion from activated macrophages and intestinal epithelial cells. However, only antibody-loaded PLGA-PEG2k nanoparticles were able to alleviate the experimental acute colitis in mice demonstrated by improved colon weight/length ratio, histological score, and reduced tissue-associated myeloperoxidase activity and expression of proinflammatory cytokine TNF-α levels compared with the control group. The results suggest that despite having no significant differences in the in vitro cell-based assays, PEG chain length has a significant impact on the in vivo performance of the mucus penetrating nanoparticles. Overall, PLGA-PEG2k nanoparticles were presented as a promising oral delivery system for targeted antibody delivery to treat inflammatory bowel disease. STATEMENT OF SIGNIFICANCE: There is an unmet therapeutic need for oral drug delivery systems for safe and effective antibody therapy of inflammatory bowel disease. Therefore, we have developed PEGylated PLGA-based nanoparticulate drug delivery systems for oral targeted delivery of anti-TNF-α antibody as a potential alternative treatment strategy. The PEG chain length did not affect encapsulation efficiency or interaction with mucin in vitro but resulted in differences in in vitro release profile and in vivo efficacy study. We demonstrated the superiority of anti-TNF-α mAb-PLGA-PEG2k over mAb-PLGA-PEG5k nanoparticles to effectively exhibit anti-inflammatory responses in an acute murine colitis model. These nanoparticle-based formulations may be adjusted to encapsulate other drugs that could be applied to a number of disorders at different mucosal surfaces.

Solid lipid nanocarriers diffuse effectively through mucus and enter intestinal cells - but where is my peptide?

Peptides are therapeutic molecules with high potential to treat a wide variety of diseases. They are large hydrophilic compounds for which absorption is limited by the intestinal epithelial border covered by mucus. This study aimed to evaluate the potential of Hydrophobic Ion Pairing combined with Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) to improve peptide transport across the intestinal border using Caco-2 cell monolayers (enterocyte-like model) and Caco-2/HT29-MTX co-cultured monolayers (mucin-secreting model). A Hydrophobic Ion Pair (HIP) was formed between Leuprolide (LEU), a model peptide, and sodium docusate. The marked increase in peptide lipophilicity enabled high encapsulation efficiencies in both NLC (84%) and SLN (85%). After co-incubation with the nanoparticles, confocal microscopy images of the cell monolayers demonstrated particles internalization and ability to cross mucus. Flow cytometry measurements confirmed that 82% of incubated SLN and 99% of NLC were internalized by Caco-2 cells. However, LEU transport across cell monolayers was not improved by the nanocarriers. Indeed, combination of particles platelet-shape and HIP low stability in the transport medium led to LEU burst release in this environment. Improvement of peptide lipidization should maintain encapsulation and enable benefit from nanocarriers enhanced intestinal transport.

Oral delivery of oleuropein-loaded lipid nanocarriers alleviates inflammation and oxidative stress in acute colitis.

Inflammation and oxidative stress pathways have emerged as novel targets in the management of inflammatory bowel diseases (IBD). Targeting the drug to the inflamed colon remains a challenge. Nanostructured lipid carriers (NLCs) have been reported to accumulate in inflamed colonic mucosa. The antioxidant/antiinflamatory polyphenol oleuropein (OLE) was loaded in NLCs (NLC-OLE). NLC-OLE showed to be more effective in decreasing the TNF-α secretion and intracellular reactive oxygen species (ROS) by activated macrophages (J774) compared to the conventional form of OLE. OLE efficacy was preserved within NLC-OLE ameliorating inflammation in a murine model of acute colitis: reduced levels of TNF-α and IL-6, decreased neutrophil infiltration and improved histopathology of the colon were reported. In addition, NLC-OLE enhanced the ROS scavenging activity of OLE in the colon after oral administration. These data suggest that the proposed NLC-OLE could be a promising drug delivery system for OLE in IBD treatment.

Targeted nanoparticles towards increased L cell stimulation as a strategy to improve oral peptide delivery in incretin-based diabetes treatment.

The delivery of therapeutic peptides via the oral route remains one of biggest challenges in the pharmaceutical industry. Recently, we have described an alternative improved drug delivery system for peptide delivery via the oral route, consisting of a lipidic nanocapsule. Despite the striking effects observed, it is still essential to develop strategies to strengthen the nanocarriers' glucagon-like peptide-1 (GLP-1) secretory effect of the nanocarrier and/or prolong its antidiabetic effect in vivo to facilitate its translation into the clinic. For this purpose, we developed and compared different fatty acid-targeted lipid and polymeric nanoparticles and evaluated the L cell stimulation induced by the nanocarriers in murine L cells in vitro and in normal healthy mice in vivo. We further examined the antidiabetic effect in vivo in an obese/diabetic mouse model induced by high-fat diet feeding and examined the effect of the oral administration frequency. Among the tested nanocarriers, only lipid-based nanocarriers that were surface-modified with DSPE-PEG2000 on the surface were able to significantly strengthen the biological effect of the nanocarriers. They increased endogenous GLP-1 levels up to 8-fold in vivo in normoglycemic mice. Moreover, they effectively prolonged the in vivo antidiabetic effect by normalizing the plasma glucose levels in obese/diabetic mice following long-term treatment (one month). Ultimately, the targeted nanocarriers were as effective when the administration frequency was reduced from once daily to once every other day.

Overcoming the intestinal barrier: A look into targeting approaches for improved oral drug delivery systems.

Oral drug administration is one of the most preferred and simplest routes among both patients and formulation scientists. Nevertheless, orally delivery of some of the most widely used therapeutic agents (e.g., anticancer drugs, peptides, proteins and vaccines) is still a major challenge due to the limited oral bioavailability associated with them. The poor oral bioavailability of such drugs is attributed to one or many factors, such as poor aqueous solubility, poor permeability, and enzymatic degradation. Various technological strategies (such as permeation enhancers, prodrugs and nanocarriers) have been developed to enhance the bioavailability of these drugs after oral administration. Among the different approaches, advanced and innovative drug delivery systems, especially targeting-based strategies, have garnered tremendous attention. Furthermore, the presence of numerous types of cells and solute carrier transporters throughout the gastrointestinal tract represents numerous potential targeting sites for successful oral delivery that have not yet been exploited for their full potential. This review describes different targeting strategies towards different targeting sites in the gastrointestinal tract. Additionally, exciting improvements in oral drug delivery systems with different targeting strategies (e.g., M cells for oral vaccination and L cells for type 2 diabetes mellitus) are also discussed.

A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation.

The specialized microfold cells (M cells) in the follicle-associated epithelium (FAE) of intestinal Peyer's patches serve as antigen-sampling cells of the intestinal innate immune system. Unlike 'classical' enterocytes, they are able to translocate diverse particulates without digesting them. They act as pathways for microorganism invasion and mediate food tolerance by transcellular transport of intestinal microbiota and antigens. Their ability to transcytose intact particles can be used to develop oral drug delivery and oral immunization strategies. This protocol describes a reproducible and versatile human M-cell-like in vitro model. This model can be exploited to evaluate M-cell transport of microparticles and nanoparticles for protein, drug or vaccine delivery and to study bacterial adherence and translocation across M cells. The inverted in vitro M-cell model consists of three main steps. First, Caco-2 cells are seeded at the apical side of the inserts. Second, the inserts are inverted and B lymphocytes are seeded at the basolateral side of the inserts. Third, the conversion to M cells is assessed. Although various M-cell culture systems exist, this model provides several advantages over the rest: (i) it is based on coculture with well-established differentiated human cell lines; (ii) it is reproducible under the conditions described herein; (iii) it can be easily mastered; and (iv) it does not require the isolation of primary cells or the use of animals. The protocol requires skills in cell culture and microscopy analysis. The model is obtained after 3 weeks, and transport experiments across the differentiated model can be carried out over periods of up to 10 h.

The interaction of protamine nanocapsules with the intestinal epithelium: A mechanistic approach.

Single-layer protamine and double layer polysialic acid (PSA)/protamine nanocapsules (NCs) were designed in order to be used as carriers to facilitate the transport of macromolecules across the intestinal epithelium. The rational for the design of these NCs was based on that protamine is a non-toxic yet potent cell-penetrating peptide, capable of translocating protein cargos through cell membranes, while PSA is a low molecular weight polysaccharide used to enhance the stability of macromolecules and nanocarriers. The aim of this work was to study in vitro the mechanism of interaction of these NCs with different intestinal cell models (Caco-2, Caco-2/Raji mimicking follicle associated epithelium and Caco-2/HT29-MTX to study the effect of mucus). For this, a fluorescent marker, TAMRA was covalently linked to protamine. The interaction and transport of the NCs with the Caco-2 cells was found to be concentration, temperature and size dependent. In all cases, the double layer PSA-protamine NCs exhibited a significantly higher transport compared to protamine NCs. On the other hand, the transport of the NCs was significantly higher in the co-culture (Caco-2/Raji monolayer) compared to the monoculture model (Caco-2 monolayer), implying that M cells are involved in the transport of these nanosystems. The formulations, administered intra-jejunally to healthy rats (4h fasting) resulted in a moderate reduction of the glucose levels (20% reduction), which lasted for up to 4h. This work raises prospects that protamine-based nanocapsules may have the potential as oral peptide delivery nanocarriers.

A comparative study of curcumin-loaded lipid-based nanocarriers in the treatment of inflammatory bowel disease.

Selective drug delivery to inflamed tissues is of widespread interest for the treatment of inflammatory bowel disease (IBD). Because a lack of physiological lipids has been described in patients suffering IBD, and some lipids present immunomodulatory properties, we hypothesize that the combination of lipids and anti-inflammatory drugs together within a nanocarrier may be a valuable strategy for overcoming IBD. In the present study, we investigated and compared the in vitro and in vivo efficacy of three lipid-based nanocarriers containing curcumin (CC) as an anti-inflammatory drug for treating IBD in a murine DSS-induced colitis model. These nanocarriers included self-nanoemulsifying drug delivery systems (SNEDDS), nanostructured lipid carriers (NLC) and lipid core-shell protamine nanocapsules (NC). In vitro, a 30-fold higher CC permeability across Caco-2 cell monolayers was obtained using NC compared to SNEDDS (NC>SNEDDS>NLC and CC suspension). The CC SNEDDS and CC NLC but not the CC NC or CC suspension significantly reduced TNF-α secretion by LPS-activated macrophages (J774 cells). In vivo, only CC NLC were able to significantly decrease neutrophil infiltration and TNF-α secretion and, thus, colonic inflammation. Our results show that a higher CC permeability does not correlate with a higher efficacy in IBD treatment, which suggests that lipidic nanocarriers exhibiting increased CC retention at the intestinal site, rather than increased CC permeability are efficient treatments of IBD.

Cyclosporine A-loaded lipid nanoparticles in inflammatory bowel disease.

Cyclosporine A (CsA) is a well-known immunosuppressive agent used as rescue therapy in severe steroid-refractory ulcerative colitis (UC). However, toxicity issues associated with CsA when administered in its commercially available formulations have been reported in clinical practice. Since nanotechnology has been proposed as a promising strategy to improve safety and efficacy in the treatment of inflammatory bowel disease (IBD), the main purpose of this study was to evaluate the effect of oral administration of CsA-loaded lipid nanoparticles (LN) in the dextran sodium sulfate (DSS)-induced colitis mouse model using Sandimmune Neoral(®) as reference. The results showed that the formulations used did not decrease colon inflammation in terms of myeloperoxidase activity (MPO), tumor necrosis factor (TNF)-α expression, or histological scoring in the acute stage of the disease. However, further studies are needed in order to corroborate the efficacy of these formulations in the chronic phase of the disease.

Algal lectin binding to core (α1-6) fucosylated N-glycans: structural basis for specificity and production of recombinant protein.

We determined the specificity of BTL, a lectin from the red marine alga Bryothamnion triquetrum, toward fucosylated oligosaccharides. BTL showed a strict specificity for the core α1,6-fucosylation, which is an important marker for cancerogenesis and quality control of therapeutical antibodies. The double fucosylation α1,6 and α1,3 was also recognized, but the binding was totally abolished in the sole presence of the α1,3-fucosylation. A more detailed analysis of the specificity of BTL showed a preference for bi- and tri-antennary nonbisected N-glycans. Sialylation or fucosylation at the nonreducing end of N-glycans did not affect the recognition by the lectin. BTL displayed a strong affinity for a core α1,6-fucosylated octasaccharide with a Kd of 12 µM by titration microcalorimetry. The structural characterization of the interaction between BTL and the octasaccharide was obtained by STD-NMR. It demonstrated an extended epitope for recognition that includes the fucose residue, the distal GlcNAc and one mannose residue. Recombinant rBTL was obtained in Escherichia coli and characterized. Its binding properties for carbohydrates were studied using hemagglutination tests and glycan array analysis. rBTL was able to agglutinate rabbit erythrocytes with strong hemagglutination activity only after treatment with papain and trypsin, indicating that its ligands were not directly accessible at the cell surface. The hemagglutinating properties of rBTL confirm the correct folding and functional state of the protein. The results show BTL as a potent candidate for cancer diagnosis and as a reagent for the preparation and quality control of antibodies lacking core α1,6-fucosylated N-glycans.

pH-sensitive nanoparticles for colonic delivery of curcumin in inflammatory bowel disease.

Nano-scaled particles have been found to preferentially accumulate in inflamed regions. Local delivery of anti-inflammatory drugs loaded in nanoparticles to the inflamed colonic site is of great interest for inflammatory bowel disease (IBD) treatment. Curcumin (CC) is an anti-inflammatory local agent, which presents poor ADME properties. Hence, we evaluated, both in vitro and in vivo, the local delivery of CC using pH-sensitive polymeric nanoparticles (NPs) combining both poly(lactide-co-glycolide) acid (PLGA) and a polymethacrylate polymer (Eudragit(®) S100). CC-NPs significantly enhanced CC permeation across Caco-2 cell monolayers when compared to CC in suspension. CC-NPs significantly reduced TNF-α secretion by LPS-activated macrophages (J774 cells). In vivo, CC-NPs significantly decreased neutrophil infiltration and TNF-α secretion while maintaining the colonic structure similar to the control group in a murine DSS-induced colitis model. Our results support the use of nanoparticles made of PLGA and Eudragit(®) S100 combination for CC delivery in IBD treatment.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Budesonide-loaded nanostructured lipid carriers reduce inflammation in murine DSS-induced colitis.

The challenge for the treatment of inflammatory bowel disease (IBD) is the delivery of the drug to the site of inflammation. Because nanoparticles have the ability to accumulate in inflamed regions, the aim of the present study was to evaluate nanostructured lipid carriers (NLCs) as nanoparticulate drug delivery systems for the treatment of IBD. Budesonide (BDS) was chosen as a candidate anti-inflammatory drug. BDS-loaded NLCs (BDS-NLC) produced by high-pressure homogenization had a size of 200 nm and a negative zeta potential. BDS-NLCs reduced the TNF-α secretion by activated macrophages (J774 cells). BDS-NLCs were more active in a murine model of dextran sulfate-induced colitis when compared with Blank-NLCs or a BDS suspension: BDS-NLCs decreased neutrophil infiltration, decreased the levels of the pro-inflammatory cytokines IL-1ß and TNF-α in the colon and improved the histological scores of the colons. These data suggest that NLCs could be a promising alternative to polymeric nanoparticles as a targeted drug delivery system for IBD treatment.

The cellular machinery of Ferroplasma acidiphilum is iron-protein-dominated.

Ferroplasma is a genus of the Archaea, one of the three branches of the tree of life, and belongs to the order Thermoplasmatales (Euryarchaeota), which contains the most acidophilic microbes yet known. Ferroplasma species live in acid mine drainage, acidic pools and environments containing sulphidic ores such as pyrite and characterized by pH values of 0-2 and high concentrations of ferrous iron and other heavy metals. F. acidiphilum strain Y(T) is a chemoautotroph that grows optimally at pH 1.7 and gains energy by oxidizing ferrous iron and carbon by the fixation of carbon dioxide. Here we show that 86% of 189 investigated cellular proteins of F. acidiphilum are iron-metalloproteins. These include proteins with deduced structural, chaperone and catalytic roles, not described as iron-metalloproteins in any other organism so far investigated. The iron atoms in the proteins seem to organize and stabilize their three-dimensional structures, to act as 'iron rivets'. Analysis of proteins of the phylogenetic neighbour Picrophilus torridus and of the habitat neighbour Acidithiobacillus ferrooxidans revealed far fewer and only typical metalloproteins. F. acidiphilum therefore has a currently unique iron-protein-dominated cellular machinery and biochemical phylogeny.

Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis, and phylogenetic relationships.

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 degrees C, pH 4.5, yielded values of 26, 0.43, and 0.45 microm and 18, 660, and 1175 s(-1), respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.