ABSTRACT
Dienelactone hydrolase (DLH) is one of numerous hydrolytic enzymes with an α/ß-hydrolase fold, which catalyze the hydrolysis of dienelactone to maleylacetate. The DLHs share remarkably similar tertiary structures and a conserved arrangement of catalytic residues. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (HtDLH). The crystal structure of the HtDLH, solved at a resolution of about 1.67â¯Å, exhibits a canonical α/ß-hydrolase fold formed by eight ß-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The HtDLH forms stable homodimers in solution. Functional studies showed that HtDLH has the expected esterase activity over esters with short carbon chains, such as p-nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70⯰C. Furthermore, HtDLH maintains more than 50â¯% of its activity even after incubation at 90⯰C for 16â¯h. Interestingly, HtDLH exhibits catalytic activity towards polyethylene terephthalate (PET) monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate, as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that HtDLH has characteristic of a BHET-degrading enzyme. This work expands our understanding of enzyme families involved in PET degradation, providing novel insights for plastic biorecycling through protein engineering, which could lead to eco-friendly solutions to reduce the accumulation of plastic in landfills and natural environments.
ABSTRACT
Plastics derived from fossil fuels are used ubiquitously owing to their exceptional physicochemical characteristics. However, the extensive and short-term use of plastics has caused environmental challenges. The biotechnological plastic conversion can help address the challenges related to plastic pollution, offering sustainable alternatives that can operate using bioeconomic concepts and promote socioeconomic benefits. In this context, using soil from a plastic-contaminated landfill, two consortia were established (ConsPlastic-A and -B) displaying versatility in developing and consuming polyethylene or polyethylene terephthalate as the carbon source of nutrition. The ConsPlastic-A and -B metagenomic sequencing, taxonomic profiling, and the reconstruction of 79 draft bacterial genomes significantly expanded the knowledge of plastic-degrading microorganisms and enzymes, disclosing novel taxonomic groups associated with polymer degradation. The microbial consortium was utilized to obtain a novel Pseudomonas putida strain (BR4), presenting a striking metabolic arsenal for aromatic compound degradation and assimilation, confirmed by genomic analyses. The BR4 displays the inherent capacity to degrade polyethylene terephthalate (PET) and produce polyhydroxybutyrate (PHB) containing hydroxyvalerate (HV) units that contribute to enhanced copolymer properties, such as increased flexibility and resistance to breakage, compared with pure PHB. Therefore, BR4 is a promising strain for developing a bioconsolidated plastic depolymerization and upcycling process. Collectively, our study provides insights that may extend beyond the artificial ecosystems established during our experiments and supports future strategies for effectively decomposing and valorizing plastic waste. Furthermore, the functional genomic analysis described herein serves as a valuable guide for elucidating the genetic potential of microbial communities and microorganisms in plastic deconstruction and upcycling.
Subject(s)
Biodegradation, Environmental , Microbiota , Plastics , Plastics/metabolism , Soil Microbiology , Polyethylene Terephthalates/metabolism , Soil Pollutants/metabolism , Polymers/metabolism , Bacteria/metabolism , Bacteria/genetics , Biodegradable Plastics/metabolism , Microbial Consortia , Pseudomonas putida/metabolism , Pseudomonas putida/geneticsABSTRACT
Staphylococcus aureus is a pathogen widely involved in wound infection due to its ability to release several virulence factors that impair the skin healing process, as well as its mechanism of drug resistance. Herein, sodium alginate and chitosan were combined to produce a hydrogel for topical delivery of neomycin to combat S. aureus associated with skin complications. The hydrogel was formulated by combining sodium alginate (50 mg/mL) and chitosan (50 mg/mL) solutions in a ratio of 9:1 (HBase). Neomycin was added to HBase to achieve a concentration of 0.4 mg/mL (HNeo). The incorporation of neomycin into the product was confirmed by scanning electron microscopy, FTIR and TGA analysis. The hydrogels produced are homogeneous, have a high swelling capacity, and show biocompatibility using erythrocytes and fibroblasts as models. The formulations showed physicochemical and pharmacological stability for 60 days at 4 ± 2 °C. HNeo totally inhibited the growth of S. aureus after 4 h. The antimicrobial effects were confirmed using ex vivo (porcine skin) and in vivo (murine) wound infection models. Furthermore, the HNeo-treated mice showed lower severity scores than those treated with HBase. Taken together, the obtained results present a new low-cost bioproduct with promising applications in treating infected wounds.
Subject(s)
Alginates , Anti-Bacterial Agents , Chitosan , Hydrogels , Neomycin , Staphylococcus aureus , Chitosan/chemistry , Chitosan/pharmacology , Alginates/chemistry , Alginates/pharmacology , Hydrogels/chemistry , Hydrogels/pharmacology , Staphylococcus aureus/drug effects , Animals , Mice , Neomycin/pharmacology , Neomycin/chemistry , Neomycin/administration & dosage , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Staphylococcal Infections/drug therapy , Staphylococcal Skin Infections/drug therapy , Staphylococcal Skin Infections/microbiology , Staphylococcal Skin Infections/pathology , Drug Carriers/chemistry , Skin/drug effects , Skin/microbiologyABSTRACT
Cellulases from anaerobic fungi are enzymes less-studied biochemically and structurally than cellulases from bacteria and aerobic fungi. Currently, only thirteen GH5 cellulases from anaerobic fungi were biochemically characterized and two crystal structures were reported. In this context, here, we report the functional and biophysical characterization of a novel multi-modular cellulosomal GH5 endoglucanase from the anaerobic gut fungus Piromyces finnis (named here PfGH5). Multiple sequences alignments indicate that PfGH5 is composed of a GH5 catalytic domain and a CBM1 carbohydrate-binding module connected through a CBM10 dockerin module. Our results showed that PfGH5 is an endoglucanase from anaerobic fungus with a large spectrum of activity. PfGH5 exhibited preference for hydrolysis of oat ß-glucan, followed by galactomannan, carboxymethyl cellulose, mannan, lichenan and barley ß-glucan, therefore displaying multi-functionality. For oat ß-glucan, PfGH5 reaches its optimum enzymatic activity at 40 °C and pH 5.5, with Km of 7.1 µM. Ion exchange chromatography analyzes revealed the production of oligosaccharides with a wide degree of polymerization indicated that PfGH5 has endoglucanase activity. The ability to bind and cleave different types of carbohydrates evidence the potential of PfGH5 for use in biotechnology and provide a useful basis for future investigation and application of new anaerobic fungi enzymes.
Subject(s)
Cellulase , Cellulases , Cellulase/chemistry , Anaerobiosis , FungiABSTRACT
Lectins are carbohydrate-binding proteins belonging to the Leguminosae family. In this family stand out proteins extracted from species belonging to Diocleinae subtribe, which includes, for example, the seed lectin from Dioclea violacea (DVL) and the jack bean lectin Concanavalin A (ConA). Here, we report the photosynthesis of silver/silver chloride nanoparticles (NPs) assisted by ConA and DVL. The syntheses were simple processes using a green-chemistry approach. Under electron microscopy, NPs heterogeneous in size, nearly spherical and covered by a thin lectin corona, were observed. Both NPs assisted by lectins were capable to cause strong rabbit erythrocytes agglutination with the same titers of hemagglutinating activities. These results indicate that both lectins maintained their biological activities even after association with the NPs and therefore are able to interact with biological membrane carbohydrates. However, for rabbit erythrocytes treated with proteolytic enzymes were observed different titers of hemagglutinating activities, suggesting differences in the spatial arrangement of the lectins on the surface of the NPs. This study provides evidences that these hybrid lectin-coated silver/silver chloride NPs can be used for selective recognition and interaction with membrane carbohydrates and others biotechnological applications.
Subject(s)
Lectins , Plant Lectins , Animals , Rabbits , Lectins/chemistry , Plant Lectins/pharmacology , Plant Lectins/chemistry , Plant Lectins/metabolism , Silver/pharmacology , Carbohydrates/chemistry , PhotosynthesisABSTRACT
The ferulic acid (FA) represents a high-value molecule with applications in the cosmetic and pharmaceutical industries. This aromatic molecule is derived from lignin and can be enzymatically converted in other commercially interesting molecules, such as vanillin and bioplastics. This process starts with a common step of FA activation via CoA-thioesterification, catalyzed by feruloyl-CoA synthetases. Therefore, here, we report the successfully expression, purification as well as the initial structural and biochemical characterization of a stable, correctly folded, and catalytically active bacterial feruloyl-CoA synthase (here named FCS3) isolated from a lignin-degrading microbial consortium. The purification of recombinant FCS3 to near homogeneity was achieved using affinity chromatography. The FCS3 structure is composed of a mixture of α and ß secondary structures and most likely forms stable homodimers in solution. The FCS3 presented a notable structural stability at alkaline pH values and it was able to convert FA and coenzyme A (CoA) into feruloyl-CoA complex at room temperature. This study should provide a useful basis for future biotechnological applications of FCS3, especially in the field of conversion of lignin-derived FA into high value compounds.
Subject(s)
Benzaldehydes , Lignin , Acyl Coenzyme A/metabolism , Benzaldehydes/metabolism , Coumaric Acids/metabolism , Lignin/metabolismABSTRACT
Deciphering how enzymes interact, modify, and recognize carbohydrates has long been a topic of interest in academic, pharmaceutical, and industrial research. Carbohydrate-binding modules (CBMs) are noncatalytic globular protein domains attached to carbohydrate-active enzymes that strengthen enzyme affinity to substrates and increase enzymatic efficiency via targeting and proximity effects. CBMs are considered auspicious for various biotechnological purposes in textile, food, and feed industries, representing valuable tools in basic science research and biomedicine. Here, we present the first crystallographic structure of a CBM8 family member (CBM8), DdCBM8, from the slime mold Dictyostelium discoideum, which was identified attached to an endo-ß-1,4-glucanase (glycoside hydrolase family 9). We show that the planar carbohydrate-binding site of DdCBM8, composed of aromatic residues, is similar to type A CBMs that are specific for crystalline (multichain) polysaccharides. Accordingly, pull-down assays indicated that DdCBM8 was able to bind insoluble forms of cellulose. However, affinity gel electrophoresis demonstrated that DdCBM8 also bound to soluble (single chain) polysaccharides, especially glucomannan, similar to type B CBMs, although it had no apparent affinity for oligosaccharides. Therefore, the structural characteristics and broad specificity of DdCBM8 represent exceptions to the canonical CBM classification. In addition, mutational analysis identified specific amino acid residues involved in ligand recognition, which are conserved throughout the CBM8 family. This advancement in the structural and functional characterization of CBMs contributes to our understanding of carbohydrate-active enzymes and protein-carbohydrate interactions, pushing forward protein engineering strategies and enhancing the potential biotechnological applications of glycoside hydrolase accessory modules.
Subject(s)
Dictyostelium , Carbohydrates/chemistry , Crystallography, X-Ray , Dictyostelium/metabolism , Glucans/metabolism , Glycoside Hydrolases , Ligands , Polysaccharides/metabolismABSTRACT
The biocatalytic production of fuels and chemicals from plant biomass represents an attractive alternative to fossil fuel-based refineries. In this context, the mining and characterization of novel biocatalysts can promote disruptive innovation opportunities in the field of lignocellulose conversion and valorization. In the present work, we conducted the biochemical and structural characterization of two novel hydroxycinnamic acid catabolic enzymes, isolated from a lignin-degrading microbial consortium, a feruloyl-CoA synthetase, and a feruloyl-CoA hydratase-lyase, named LM-FCS2 and LM-FCHL2, respectively. Besides establishing the homology model structures for novel FCS and FCHL members with unique characteristics, the enzymes presented interesting biochemical features: LM-FCS2 showed stability in alkaline pHs and was able to convert a wide array of p-hydroxycinnamic acids to their respective CoA-thioesters, including sinapic acid; LM-FCHL2 efficiently converted feruloyl-CoA and p-coumaroyl-CoA into vanillin and 4-hydroxybenzaldehyde, respectively, and could produce vanillin directly from ferulic acid. The coupled reaction of LM-FCS2 and LM-FCHL2 produced vanillin, not only from commercial ferulic acid but also from a crude lignocellulosic hydrolysate. Collectively, this work illuminates the structure and function of two critical enzymes involved in converting ferulic acid into high-value molecules, thus providing valuable concepts applied to the development of plant biomass biorefineries. KEY POINTS: ⢠Comprehensive characterization of feruloyl-CoA synthetase from metagenomic origin. ⢠Novel low-resolution structures of hydroxycinnamate catabolic enzymes. ⢠Production of vanillin via enzymatic reaction using lignocellulosic hydrolysates.
Subject(s)
Lignin , Metagenome , Escherichia coli/genetics , Hyperlipidemia, Familial Combined , Lignin/metabolism , SoilABSTRACT
Lytic polysaccharide monooxygenases (LPMOs) are metalloenzymes that cleave structural polysaccharides through an oxidative mechanism. The enzymatic activity of LPMOs relies on the presence of a Cu2+ histidine-brace motif in their flat catalytic surface. Upon reduction by an external electron donor and in the presence of its co-substrates, O2 or H2O2, LPMOs can generate reactive oxygen species to oxidize the substrates. Fungal and bacterial LPMOs are involved in the catabolism of polysaccharides, such as chitin, cellulose, and hemicelluloses, and virulence mechanisms. Based on the reports on the discovery of LPMOs from the family AA15 in termites, firebrats, and flies, the functional role of the LPMO in the biosphere could expand, as these enzymes may be correlated with chitin remodeling and molting in insects. However, there is limited knowledge of AA15 LPMOs due to difficulties in recombinant expression of soluble proteins and purification protocols. In this study, we describe a protocol for the cloning, expression, and purification of insect AA15 LPMOs from Arthropoda, mainly from termites, followed by the expression and purification of an AA15 LPMO from the silkworm Bombyx mori, which contains a relatively high number of disulfide bonds. We also report the recombinant expression and purification of a protein with homology to AA15 family from the western European honeybee Apis mellifera, an LPMO-like enzyme lacking the canonical histidine brace. Therefore, this work can support future studies concerning the role of LPMOs in the biology of insects and inspire molecular entomologists and insect biochemists in conducting activities in this field.
Subject(s)
Bees/genetics , Escherichia coli , Gene Expression , Insect Proteins , Mixed Function Oxygenases , Animals , Bees/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Insect Proteins/biosynthesis , Insect Proteins/chemistry , Insect Proteins/genetics , Insect Proteins/isolation & purification , Mixed Function Oxygenases/biosynthesis , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Fructooligosaccharides (FOSs) are polymers of fructose with a prebiotic activity because of their production and fermentation by bacteria that inhabit the gastrointestinal tract and are widely used in the industry and new functional foods. Lactobacillus gasseri stands out as an important homofermentative microorganism related to FOS production, and its potential applications in the industry are undeniable. In this study, we report the production and characterization of a sucrose-6-phosphate hydrolase from L. gasseri belonging to the GH32 family. Apo-LgAs32 and LgAs32 complexed with ß-d-fructose structures were determined at a resolution of 1.94 and 1.84 Å, respectively. The production of FOS, fructans, 1-kestose, and nystose by the recombinant LgAs32, using sucrose as a substrate, shown in this study is very promising. When compared to its homologous enzyme from Lactobacillus reuteri, the production of 1-kestose by LgAs32 is increased; thus, LgAs32 can be considered as an alternative in fructan production and other industrial applications.
Subject(s)
Hexosyltransferases , Lactobacillus gasseri , Food Industry , Fructans , Hexosyltransferases/genetics , Oligosaccharides , Sucrose/analogs & derivatives , Sugar Phosphates , beta-Fructofuranosidase/geneticsABSTRACT
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which catalyze the oxidative cleavage of polysaccharides. LPMOs belonging to family 15 in the Auxiliary Activity (AA) class from the Carbohydrate-Active Enzyme database are found widespread across the Tree of Life, including viruses, algae, oomycetes and animals. Recently, two AA15s from the firebrat Thermobia domestica were reported to have oxidative activity, one towards cellulose or chitin and the other towards chitin, signalling that AA15 LPMOs from insects potentially have different biochemical functions. Herein, we report the identification and characterization of two family AA15 members from the lower termite Coptotermes gestroi. Addition of Cu(II) to CgAA15a or CgAA15b had a thermostabilizing effect on both. Using ascorbate and O2 as co-substrates, CgAA15a and CgAA15b were able to oxidize chitin, but showed no activity on celluloses, xylan, xyloglucan and starch. Structural models indicate that the LPMOs from C. gestroi (CgAA15a/CgAA15b) have a similar fold but exhibit key differences in the catalytic site residues when compared to the cellulose/chitin-active LPMO from T. domestica (TdAA15a), especially the presence of a non-coordinating phenylalanine nearby the Cu ion in CgAA15a/b, which appears as a tyrosine in the active site of TdAA15a. Despite the overall similarity in protein folds, however, mutation of the active site phenylalanine in CgAA15a to a tyrosine did not expanded the enzymatic specificity from chitin to cellulose. Our data show that CgAA15a/b enzymes are likely not involved in lignocellulose digestion but might play a role in termite developmental processes as well as on chitin and nitrogen metabolisms.
Subject(s)
Copper/chemistry , Insect Proteins/chemistry , Isoptera/enzymology , Mixed Function Oxygenases/chemistry , Models, Molecular , Animals , Copper/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Isoptera/genetics , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolismABSTRACT
rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential. Besides that, rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of rBmTI-A was modeled. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism spectroscopy data corroborated the secondary structure found by the homology modelling. Also, in circular dichroism data it was shown a thermostability of rBmTI-A until approximately 70 °C, corroborated by inhibitory assays toward trypsin.
Subject(s)
Arthropod Proteins/chemistry , Molecular Dynamics Simulation , Rhipicephalus/chemistry , Serine Proteinase Inhibitors/chemistry , Animals , Arthropod Proteins/genetics , Arthropod Proteins/pharmacology , Disease Models, Animal , Humans , Leukocyte Elastase/antagonists & inhibitors , Leukocyte Elastase/metabolism , Mice , Protein Stability , Pulmonary Emphysema/drug therapy , Pulmonary Emphysema/metabolism , Pulmonary Emphysema/pathology , Rhipicephalus/genetics , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/pharmacologyABSTRACT
The lectins are carbohydrate-binding proteins that are highly specific to sugar groups associated to other molecules. In addition to interacting with carbohydrates, a number of studies have reported the ability of these proteins to modulate the activity of several antibiotics against multidrug-resistant (MDR) strains. In this study, we report the enhanced antibacterial activity of the gentamicin against MDR strains when complexed with a lectin from Canavalia ensiformis seeds (ConA). Hemagglutination activity test and intrinsic fluorescence spectroscopy revealed that the gentamicin can interact with ConA most likely via the carbohydrate recognition domain (CRD) with binding constant (Kb) value estimated of (0.44 ± 0.04) x 104 M-1. Furthermore, the minimum inhibitory concentrations (MIC) obtained for ConA against all strains studied were not clinically relevant (MIC ≥ 1024 µg/mL). However, when ConA was combined with gentamicin, a significant increase in antibiotic activity was observed against Staphylococcus aureus and Escherichia coli. The present study showed that ConA has an affinity for gentamicin and modulates its activity against MDR strains. These results indicate that ConA improves gentamicin performance and is a promising candidate for structure/function analyses.
Subject(s)
Canavalia , Gentamicins , Anti-Bacterial Agents/pharmacology , Gentamicins/pharmacology , Lectins , Microbial Sensitivity TestsABSTRACT
The fungus Thermothielavioides terrestris plays an important role in the global carbon cycle with enzymes capable of degrading polysaccharides from biomass, therefore an attractive source of proteins to be investigated and understood. From cloning to a three-dimensional structure, we foster a deeper characterization of an α-Ê-arabinofuranosidase, a glycoside hydrolase from the family 62 (TtAbf62), responsible to release arabinofuranose from non-reducing ends of polysaccharides. TtAbf62 was tested with synthetic (pNP-Araf) and polymeric substrates (arabinan and arabinoxylan), showing optimal temperature and pH (for pNP-Araf) of 30 °C and 4.5-5.0, respectively. Kinetic parameters revealed different specific activity for the three substrates, with a higher affinity for pNP-Araf (KM: 4 ± 1 mM). The hydrolyzing activity of TtAbf62 on sugarcane bagasse suggests high efficiency in the decomposition of arabinoxylan, abundant hemicellulose presented in the sugarcane cell wall. The crystal packing of TtAbf62 reveals an exquisite domain swapping, located at the supramolecular arrangement through a disulfide bond. All crystallographic behaviors go against its monomeric state in solution, indicating a crystal-induced artifact. Structural information will form the basis for further studies aiming the development of optimized enzymatic properties to be used in biotechnological applications.
Subject(s)
Ascomycota/enzymology , Glycoside Hydrolases/chemistry , Models, Molecular , Protein Conformation , Protein Interaction Domains and Motifs , Biochemical Phenomena , Catalysis , Glycoside Hydrolases/metabolism , Kinetics , Protein Binding , Recombinant Proteins , Spectrum Analysis , Structure-Activity RelationshipABSTRACT
Mycobacterium abscessus subsp. massiliense (Mycma) belongs to the Mycobacterium abscessus complex and is a rapidly growing non-tuberculous mycobacterium. The chronic pulmonary, skin, and soft tissue infections that it causes may be difficult to treat due to its intrinsic resistance to the commonly used antimicrobial drugs, making it a serious world public health problem. Iron is an essential nutrient for the growth of microorganisms; nonetheless, it can be toxic when in excess. Thus, bacteria require an iron homeostasis mechanism to succeed in different environments. DNA-binding proteins from starved cells (Dps) are miniferritins with the property to act as additional iron storage proteins but also can bind to DNA, protecting it against hydroxyl radical. Annotation of the Mycma genome revealed the gene mycma_03135 with 79% sequential identity when compared to MSMEG_3242 gene from M. smegmatis mc2 155, which codifies for a known Dps. Recombinant Dps from M. abscessus (rMaDps) was produced in Escherichia coli, purified in soluble form and shown to form high mass oligomers in solution with ferroxidase activity, DNA binding, and protection against damage. The expression of the mycma_03135 gene was induced during Mycma growth in the presence of hydrogen peroxide (H2O2). Additionally, the expression of rMaDps by E. coli conferred greater resistance to H2O2. Thus, this study is the first to identify and characterize a Dps from M. abscessus. KEY POINTS: Mycobacterium abscessus subsp. massiliense express a miniferritin protein (Dps). Mycma Dps binds to DNA and protects against oxidative stress.
Subject(s)
Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Mycobacterium abscessus/genetics , Mycobacterium abscessus/metabolism , Stress, Physiological , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Genome, Bacterial , Hydrogen Peroxide/pharmacology , Mycobacterium abscessus/drug effects , Sequence Analysis, DNAABSTRACT
The endo-ß-1,4-mannanase from the hyperthermostable bacterium Thermotoga petrophila (TpMan) is an enzyme that catalyzes the hydrolysis of mannan and heteromannan polysaccharides. Of the three domains that comprise TpMan, the N-terminal GH5 catalytic domain and the C-terminal carbohydrate-binding domain are connected through a central ancillary domain of unknown structure and function. In this study, we report the partial crystal structure of the TpMan at 1.45 Å resolution, so far, the first modular hyperthermostable endo-ß-1,4-mannanase structure determined. The structure exhibits two domains, a (ß/α)8-barrel GH5 catalytic domain connected via a linker to the central domain with an immunoglobulin-like ß-sandwich fold formed of seven ß-strands. Functional analysis showed that whereas the immunoglobulin-like domain does not have the carbohydrate-binding function, it stacks on the GH5 catalytic domain acting as a thermostabilizing domain and allowing operation at hyperthermophilic conditions. The carbohydrate-binding domain is absent in the crystal structure most likely due to its high flexibility around the immunoglobulin-like domain which may act also as a pivot. These results represent new structural and functional information useful on biotechnological applications for biofuel and food industries.
Subject(s)
Bacteria/chemistry , Bacterial Proteins/chemistry , Immunoglobulin Domains , Mannans/chemistry , Mannosidases/chemistry , Bacteria/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Hydrolysis , Hydrophobic and Hydrophilic Interactions , Mannans/metabolism , Mannosidases/genetics , Mannosidases/metabolism , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , ThermotogaABSTRACT
Ecotin, a serine peptidase inhibitor (ISP), discovered in Escherichia coli, inhibit a wide range of trypsin-like serine peptidases, protecting microorganisms from the host's immune response. In eukaryotes, ISPs encoding genes were found only in Trypanosomatidae protozoa, including the genus Trypanosoma, which harbors Trypanosoma cruzi, the ethiological agent of Chagas' disease. T. cruzi encodes the ISP2 Trypanosomatidae orthologous, which in Leishmania species present inhibitory activity on mammalian proteases from S1A family suggesting its role in vertebrate-host-parasite interactions. In this study, the structural and biochemical characterization of the recombinant T. cruzi ISP2 (rTcISP2), produced in E. coli was purified in soluble form and analyzed by circular dichroism, fluorescence spectroscopy, native electrophoresis, dynamic light scattering, low X-ray scattering and homology modeling. The obtained data revealed that rTcISP2 was biologically active and forms homodimers in solution. Furthermore, inhibitory activity of rTcISP2 against human neutrophil elastase (HNE) is the highest among ISP2 orthologous from bacteria and trypanosomatids. The role of NE to control T. cruzi parasites through modulation of cellular and humoral innate immune responses in vertebrate hosts, make TcISP2 a key molecular component for parasite infection efficiency, providing a useful basis for investigation of host-parasite interactions and the potential of TcISP2 for biotechnological applications.
Subject(s)
Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/drug effects , Amino Acid Sequence , Chagas Disease/drug therapy , Chagas Disease/parasitology , Enzyme Activation/drug effects , Gene Expression , Hydrogen-Ion Concentration , Recombinant Proteins , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/isolation & purification , Structure-Activity Relationship , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/geneticsABSTRACT
In the context of increasing demand for renewable alternatives of fuels and chemicals, the valorization of lignin emerges as a value-adding strategy in biorefineries and an alternative to petroleum-derived molecules. One of the compounds derived from lignin is ferulic acid (FA), which can be converted into valuable molecules such as vanillin. In microorganisms, FA biotransformation into vanillin can occur via a two-step reaction catalyzed by the sequential activity of a feruloyl-CoA synthetase (FCS) and an feruloyl-CoA hydratase-lyase (FCHL), which could be exploited industrially. In this study, a prokaryotic FCHL derived from a lignin-degrading microbial consortium (named LM-FCHL) was cloned, successfully expressed in soluble form and purified. The crystal structure was solved and refined at 2.1 Å resolution. The LM-FCHL is a hexamer composed of a dimer of trimers, which showed to be quite stable under extreme pH conditions. Finally, small angle X-ray scattering corroborates the hexameric state in solution and indicates flexibility in the protein structure. The present study contributes to the field of lignin valorization to valuable molecules by establishing the biophysical and structural characterization for a novel FCHL member of unique characteristics.
Subject(s)
Benzaldehydes/metabolism , Coumaric Acids/metabolism , Hydro-Lyases/chemistry , Lignin/metabolism , Acyl Coenzyme A/metabolism , Hydro-Lyases/metabolism , Hydrogen-Ion Concentration , Microbial Consortia , Models, Molecular , Protein MultimerizationABSTRACT
Gentamicin is an aminoglycoside antibiotic used to treat infections of various origins. In the last few decades, the constant use of gentamicin has resulted in increased bacterial resistance and nephrotoxicity in some cases. In this study, we examined the ability of Dioclea violacea lectin (DVL) in modulate the antimicrobial activity of gentamicin and reduce the nephrotoxicity induced by this drug. The minimum inhibitory concentration (MIC) obtained for DVL against all strains studied was not clinically relevant (MIC ≥ 1024 µg/mL). However, when DVL was combined with gentamicin, a significant increase in antibiotic action was observed against Staphylococcus aureus and Escherichia coli. DVL also reduced antibiotic tolerance in S. aureus during 10 days of continuous treatment. In addition, DVL presented a nephroprotective effect, reducing sodium excretion, N-Gal expression and urinary protein, that are important markers of glomerular and tubular injuries. Taken together, studies of inhibition of hemagglutinating activity, fluorescence spectroscopy and molecular docking revealed that gentamicin can interact with DVL via the carbohydrate recognition domain (CRD), suggesting that the results obtained in this study may be directly related to the interaction of DVL-gentamicin and with the ability of the lectin to interact with glycans present in the cells of the peritoneum.
Subject(s)
Anti-Bacterial Agents/pharmacology , Dioclea/chemistry , Drug Resistance, Multiple, Bacterial/drug effects , Gentamicins/pharmacology , Kidney/pathology , Plant Lectins/pharmacology , Protective Agents/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Gentamicins/chemistry , Hemagglutination/drug effects , Kidney/drug effects , Kidney/injuries , Kidney/physiopathology , Male , Microbial Sensitivity Tests , Molecular Docking Simulation , Plant Lectins/chemistry , Plant Lectins/isolation & purification , Rabbits , Rats, Wistar , Reactive Oxygen Species/metabolism , Spectrometry, FluorescenceABSTRACT
Enzymes are essential in many biological processes, including second-generation ethanol production. However, enzymes are one of the main expenses for the industrial process in these days. Several studies have been done to maximize cost savings, however, many processes are still economically infeasible. In this study, we report the synthesis of a suspension of lignocresol for recycling or reuse of enzymes in bioprocesses. In this way, it was performed the adsorption assays between lignocresol and ß-glucosidases from Thermotoga petrophila, belonging to the families GH1 and GH3, for the development of a lignocresol-enzyme complex. Our results show that lignocresol maintains greater adsorptive capacity for ß-glucosidases than lignin. This capacity can be explained both by its great hydrophobicity and also by electrostatic characteristics. Therefore, all these results demonstrate good adsorption of the enzymes to the lignocresol, demonstrating great potential for enzymatic recycling.