Pyrethroid Carboxylesterase PytH from Sphingobium faniae JZ-2: Structure and Catalytic Mechanism.

Carboxylesterase PytH, isolated from the pyrethroid-degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin, and bifenthrin. To elucidate the catalytic mechanism of PytH, we report here the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported α/ß-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230, and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low-activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrate binding; both lid and core domains are involved in substrate binding, and the lid domain-induced core domain movement may make the active center correctly positioned with substrates.IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household; however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. This showed that PytH belongs to the α/ß-hydrolase fold proteins with typical catalytic Ser-His-Asp triad, though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket enabled PytH to bind bigger pyrethroid family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deepen our understanding of α/ß-hydrolase members.

Interaction of Human Drug-Metabolizing CYP3A4 with Small Inhibitory Molecules.

Binding of small inhibitory compounds to human cytochrome P450 3A4 (CYP3A4) could interfere with drug metabolism and lead to drug-drug interactions, the underlying mechanism of which is not fully understood due to insufficient structural information. This study investigated the interaction of recombinant CYP3A4 with a nonspecific inhibitor metyrapone, antifungal drug fluconazole, and protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Metyrapone and fluconazole are classic type II ligands that inhibit CYP3A4 with medium strength by ligating to the heme iron, whereas PMSF, lacking the heme-ligating moiety, acts as a weak type I ligand and inhibitor of CYP3A4. High-resolution crystal structures revealed that the orientation of metyrapone is similar but not identical to that in the previously reported 1W0G model, whereas the flexible fluconazole adapts a conformer markedly different from that observed in the target CYP51 enzymes, which could explain its high potential for cross-reactivity. Besides hydrophobic and aromatic interactions with the heme and active site residues, both drugs establish water-mediated contacts that stabilize the inhibitory complexes. PMSF also binds near the catalytic center, with the phenyl group parallel to the heme. However, it does not displace the water ligand and is held in place via strong H-bonds formed by the sulfofluoride moiety with Ser119 and Arg212. Collectively, our data suggest that PMSF might have multiple binding sites and likely occupies the high-affinity site in the crystal structure. Moreover, its hydrolysis product, phenylmethanesulfonic acid, can also access and be retained in the CYP3A4 active site. Therefore, to avoid experimental artifacts, PMSF should be excluded from purification and assay solutions.

Production, purification, and characterization of a novel serine-esterase from Aspergillus westerdijkiae.

Esterases hydrolyze water soluble short chain fatty acids esters and are biotechnologically important. A strain of Aspergillus westerdijkiae isolated from cooking oil for recycling was found to secrete an esterase. The best enzyme production (19-24 U/ml of filtrate) culture conditions were stablished. The protein was purified using ammonium sulphate precipitation, dialysis, and a chromatographic step in Sephacryl S-200 HR. The 32 kDa purified protein presented an optimal temperature of 40°C, with a T50 of 48.95°C, and an optimal pH of 8.0. KM and Vmax were 638.11 µM for p-NPB and 5.47 µmol of released p-NP · min-1 · µg-1 of protein, respectively. The purified enzyme was partially active in the presence of 25% acetone. PMSF inhibited the enzyme, indicating that it is a serine hydrolase. MS enzyme peptides sequences were used to find the protein in the A. westerdijkiae sequenced genome. A structure model demonstrated that the protein is a member of the a/ß -hydrolase fold superfamily.

Characterization of thermo-solvent stable protease from Halobacillus sp. CJ4 isolated from Chott Eldjerid hypersaline lake in Tunisia.

About 110 newly isolated halophilic and halotolerant bacteria were screened for protease production. A moderately halophilic strain (CJ4), isolated from Chott Eldjerid Hypersaline lake in Tunisia, showed the highest activity on agar plate and was then selected. The biochemical and physiological characterization of the isolate along with the 16S rRNA sequence analysis placed it in the genus Halobacillus. Protease production was maximal at 120 g/L NaCl (2 M) and it started from the post-exponential phase reaching a maximum level at the early decline phase of bacterial growth. Protease activity was optimal at 0.4 M NaCl, pH 9 and 45 °C. It showed an excellent stability over wide ranges of temperatures (30-60 °C), NaCl concentrations (0-5 M), and pH values (5-10), which make it a good candidate for industrial applications at harsh conditions. Crude protease was strongly inhibited by PMSF revealing the dominance of serine proteases. Protease activity exhibited high stability in the presence of several organic solvents and detergent additives. These findings make Halobacillus sp. CJ4 protease with a great interest for many biotechnological applications at high salt or low water content such as peptide synthesis and detergent formulation.

Identification and characterization of a chymotrypsin-like serine protease from periodontal pathogen, Tannerella forsythia.

Tannerella forsythia is a bacteria associated with severe periodontal disease. This study reports identification and characterization of a membrane-associated serine protease from T. forsythia. The protease was isolated from T. forsythia membrane fractions and shown to cleave both gelatin and type I collagen. The protease was able to cleave both substrates over a wide range of pH values, however optimal cleavage occurred at pH 7.5 for gelatin and 8.0 for type I collagen. The protease was also shown to cleave both gelatin and type I collagen at the average reported temperature for the gingival sulcus however it showed a lack of thermal stability with a complete loss of activity by 60 °C. When treated with protease inhibitors the enzyme's activity could only be completely inhibited by serine protease inhibitors antipain and phenylmethanesulfonyl fluoride (PMSF). Further characterization of the protease utilized serine protease synthetic peptides. The protease cleaved N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide but not Nα-benzoyl-dl-arginine p-nitroanilide (BAPNA) or N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide indicating that the protease is a chymotrypsin-like serine protease. Since type I collagen is a major component in the gingival tissues and periodontal ligament, identification and characterization of this enzyme provides important information regarding the role of T. forsythia in periodontal disease.

Production and purification of novel thermostable alkaline protease from Anoxybacillus sp. KP1.

In this study, an extracellular novel alkaline protease (EC 3.4.21-24, 99) from a thermophilic and aerobic strain of Anoxybacillus sp. KP1 has been studied. Maximum protease activity was obtained at 50 degC at pH 9.0 after 24 hours of incubation. Among the carbon and nitrogen sources used; the optimum protease production was with soluble starch, maltose, urea and casamino acid. The enzyme was purified by ammonium sulphate precipitation and Sephadex G-75 gel chromatography. Molecular weight of purified enzyme was determined as 106 kDa by SDS-PAGE. Purified protease was stable at 50-60 °C and at pH 9.0 for 1 h. The enzyme activity was increased in the presence of Ca2+, Cu2+, Tween 80 and Triton X-100, however the enzyme activity was inhibited in the presence of Hg2+, ethylene diamine tetra acetic acid (EDTA) and H2O2. Proteolytic activity was completely inhibited by phenyl methyl sulfonyl fluoride (PMSF). The enzyme seems to be a serine alkaline protease. In the presence of detergents, the protease was clearly stable and residual activity was between 73-82%.

A Highly Stable D-Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus.

d-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic d-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested d-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain d-amino acids, with the highest specific activity toward d-valine and catalytic efficiency (kcat/Km) toward d-leucine. Substrate inhibition was observed in the case of d-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain d-amino acids, and as a scaffold to generate a novel DAO via protein engineering.

Purification and biochemical characterization of a novel protease from Penicillium digitatum - Use in bioactive peptides production.

This work reports the production of a novel serine protease enzyme (P. dig-protease) from the fungus Penicillium digitatum. The protease was purified from the culture supernatant to homogeneity using ammonium sulfate precipitation, Sephadex G-150 gel filtration and carboxymethyl-sepharose ion exchange chromatography with a 13-fold increase in specific activity. The apparent molecular weight of P.dig-protease was estimated to be 120 kDa by native high performance liquid chromatography (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single polypeptide at about 30 kDa that indicates a tetrameric protein. The proteolytic activity was inhibited by phenylmethylsulfonyl fluoride suggesting a serine-protease enzyme. P.dig-protease stability was investigated over broad range of pH, temperature, salt concentrations, surfactants and metal ions. The purified P.dig-protease was used for the production of bioactive peptides. Red scorpionfish (Scorpaena notata) muscle was hydrolyzed with P.dig-protease in order to obtain peptides with biological activities. Interestingly, the hydrolysate revealed the presence of antioxidant and angiotensin-I converting enzyme inhibitor peptides.

IgGs containing light chains of the λ- and κ- type and of all subclasses (IgG1-IgG4) from the sera of patients with systemic lupus erythematosus hydrolyze myelin basic protein.

Human myelin basic protein (hMBP)-hydrolyzing activity was recently shown to be an intrinsic property of antibodies from systemic lupus erythematosus (SLE) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from SLE patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hMBP at different optimal pH values (5.3-9.5); the pH profiles of IgG1, IgG2, IgG3 and IgG4 were unique. IgGs containing the λ-type of light chains demonstrated higher relative activities (RAs) in the hydrolysis of hMBP and its oligopeptides (OPs) than κ-IgGs. IgGs of all four subclasses were catalytically active; their RAs in the hydrolysis of hMBP increased in the following order: IgG4 < IgG2 < IgG3 < IgG1. Metal-dependent proteolytic activity of λ-IgG, IgG1, IgG2 and IgG3 was higher than their serine protease-like activity, while these activities of κ-IgG were comparable. Phenylmethylsulfonylfluoride had almost no effect on the activity of IgG4, while EDTA significantly suppressed its activity. The RAs of λ-IgG in the hydrolysis of four OPs corresponding to different cleavage sites of hMBP were remarkably higher than those for κ-IgGs. IgG1-IgG4 demonstrated different RAs and patterns of hydrolysis of these four OPs. Although combination of Ca²âº plus Mg²âº was the best in the activation of IgG1 and IgG2, IgG3 and IgG4 demonstrated the highest activity in the presence of Ca²âº plus Co²âº. The ratio of the RAs of λ-IgG, κ-IgG and IgG1-IgG4 preparations in all analyzed cases was individual for each preparation.

Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

The obligate predator Bdellovibrio bacteriovorus HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of B. bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ(Bd)). The primary structure of PhaZ(Bd) suggests that this enzyme belongs to the α/ß-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZ(Bd) has been extracellularly produced using different hypersecretor Tol-pal mutants of Escherichia coli and Pseudomonas putida as recombinant hosts. The recombinant PhaZ(Bd) has been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZ(Bd) is an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Extracellular expression and characterization of thermostable lipases, LIP8, LIP14 and LIP18, from Yarrowia lipolytica.

Two new lipases, LIP14 and LIP18, along with LIP8 from Yarrowia lipolytica MSR80 were functionally expressed as extracellular proteins with an IgG tag using Escherichia coli HB101 pEZZ18 host vector system. Each enzyme had an optimal activity at pH 7 and 40 °C and was activated by 6 mM Ca(2+) and 90 % (v/v) non-polar solvents but inhibited by 10 mM of each 1,10-phenanthraline, DTNB, PMSF and N-bromosuccinamide. All the enzymes were thermostable with t(1/2) of 52 min, 49 min and 68 min for LIP8, LIP14 and LIP18 at 80 °C, respectively. LIP18 was most thermostable among all with a high arginine: lysine ratio and proline content. All the three lipases showed a preference for oleic acid rich triacylglycerols and oils.

In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum.

A serine protease from the nematophagous fungus Monacrosporium thaumasium (NF34a) was purified, partially characterized and tested in vitro in control of the first larval stage of Angiostrongylus vasorum. NF34a grew in liquid culture medium, producing its crude extract that was purified by ion exchange chromatography. The fractions with high protease activity were collected in a pool, and elution of proteases was monitored by enzymatic assay and protein content. Purification steps were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protease activity was determined under different pH and temperature conditions, and the inhibitor effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) were assessed. In an experimental test, the infection process of NF34a on first-stage larvae of A. vasorum was investigated. A purified serine protease (Mt1) was identified, with an approximate molecular mass of 40 kDa and apparent homogeneity in SDS-PAGE, having optimal activity at pH 7.0 to 8.0 and temperature of 60°C. Mg(2+) and Zn(2+) partially inhibited the activity of Mt1 while PMSF inhibited it completely. Mt1 production was observed when NF34a was grown using first-stage larvae of A. vasorum as the only source of carbon and nitrogen. These results show that the enzyme may have a possible role in the infection process of the larvae. In the in vitro test of applicability against A. vasorum L(1), we observed a reduction in the number of larvae of 23.9% (p < 0.05) in the group treated with Mt1 compared with the control group. However, even this low reduction demonstrates that the Mt1 is important in the infection process.

Purification and characterization of a secretory lipolytic enzyme, MgLIP2, from Malassezia globosa.

Malassezia globosa is a lipid-dependent yeast that is found on the human skin and is associated with various skin disorders, including dandruff and seborrhoeic dermatitis (SD). Despite its important role in skin diseases, the molecular basis for its pathogenicity is poorly understood. The current hypothesis is that dandruff and SD are linked to fatty acid metabolism and secretory lipolytic enzymes, which hydrolyse sebaceous lipids and release irritating free fatty acids. A previous genomic analysis of M. globosa identified a family of 13 homologous genes predicted to encode secreted lipases. We have also reported that M. globosa had significantly higher extracellular lipase activity compared with other species. To identify the major secretory lipases of this yeast during its growth, we successfully purified and characterized an extracellular lipase MgLIP2. Based on MALDI-TOF MS, the peptide mass fingerprint of a tryptically digested protein MgLIP2 corresponded to ORF MGL_4054 of M. globosa. This lipase showed high esterase activity against 4-nitrophenyl palmitate and 1-naphthyl palmitate but not 1-naphthyl acetate. This enzyme had optimal activity at 30 °C and pH 5.0. Furthermore, the activity significantly increased in the presence of Triton X-100 and was partially inhibited by PMSF but was unaffected by univalent and divalent metal ions.

Addition of Phenylmethylsulfonyl Fluoride Increases the Working Lifetime of the Trout Liver S9 Substrate Depletion Assay, Resulting in Improved Detection of Low Intrinsic Clearance Rates.

The activity of a trout liver S9 substrate depletion assay has been shown to decline over time, presumably due to proteolytic degradation of biotransformation enzymes. To address this problem, assay performance was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general-purpose protease inhibitor cocktail to liver homogenization buffers and/or S9 reaction mixtures. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on clearance of phenanthrene, a model cytochrome P450 substrate, in short-term (25 or 30 min) depletion experiments but resulted in significant improvements in retention of this initial activity over time. The protease inhibitor cocktail strongly inhibited initial activity when added to homogenization buffers or reaction mixtures. Taking into consideration potential effects on liver carboxylesterases, the treatment approach determined to be optimal was addition of 10 µM PMSF to the S9 reaction mixture. Addition of 10 µM PMSF to the mixture resulted in significantly higher rates of phenanthrene clearance in 2-h incubations relative to those obtained in the absence of PMSF and a 6-fold increase in the working lifetime of the preparation. The results of a statistical power analysis suggest that by increasing the working lifetime of the assay, addition of PMSF to the reaction mixture could result in substantially improved detection of low in vitro clearance rates when compared to current practice. These findings demonstrate the value of adding PMSF to the trout S9 preparation and may have broad implications for use of this assay to support chemical bioaccumulation assessments for fish. Environ Toxicol Chem 2021;40:148-161. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Purification and characteristics of trypsin from masu salmon (Oncorhynchus masou) cultured in fresh-water.

Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl cellulose to obtain a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The molecular mass of the purified trypsin was estimated to be approximately 24,000 Da by SDS-PAGE. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and N ( alpha )-p-tosyl-L: -lysine chloromethyl ketone. Masu salmon trypsin was stabilized by calcium ion. The optimum pH of the masu salmon trypsin was around pH 8.5, and the trypsin was unstable below pH 5.0. The optimum temperature of the masu salmon trypsin was around 60 degrees C, and the trypsin was stable below 50 degrees C, like temperate-zone and tropical-zone fish trypsins. The N-terminal 20 amino acid sequence of the masu salmon trypsin was IVGGYECKAYSQPHQVSLNS, and its charged amino acid content was lower than those of trypsins from frigid-zone fish and similar to those of trypsins from temperate-zone and tropical-zone fish. In the phylogenetic tree, the masu salmon trypsin was classified into the group of the temperate-zone fish trypsin.

The fungal subtilase AsES elicits a PTI-like defence response in Arabidopsis thaliana plants independently of its enzymatic activity.

Acremonium strictum elicitor subtilisin (AsES) is a 34-kDa serine-protease secreted by the strawberry fungal pathogen A. strictum. On AsES perception, a set of defence reactions is induced, both locally and systemically, in a wide variety of plant species and against pathogens of alternative lifestyles. However, it is not clear whether AsES proteolytic activity is required for triggering a defence response or if the protein itself acts as an elicitor. To investigate the necessity of the protease activity to activate the defence response, AsES coding sequences of the wild-type gene and a mutant on the active site (S226A) were cloned and expressed in Escherichia coli. Our data show that pretreatment of Arabidopsis plants with inactive proteins, i.e. inhibited with phenylmethylsulphonyl fluoride (PMSF) and mutant, resulted in an increased systemic resistance to Botrytis cinerea and expression of defence-related genes in a temporal manner that mimics the effect already reported for the native AsES protein. The data presented in this study indicate that the defence-eliciting property exhibited by AsES is not associated with its proteolytic activity. Moreover, the enhanced expression of some immune marker genes, seedling growth inhibition and the involvement of the co-receptor BAK1 observed in plants treated with AsES suggests that AsES is being recognized as a pathogen-associated molecular pattern by a leucine-rich repeat receptor. The understanding of the mechanism of action of AsES will contribute to the development of new breeding strategies to confer durable resistance in plants.

Molecular dynamics simulations of the interaction of Mouse and Torpedo acetylcholinesterase with covalent inhibitors explain their differential reactivity: Implications for drug design.

Although the three-dimensional structures of mouse and Torpedo californica acetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas the Torpedo enzyme is effectively inhibited by benzenesulfonyl fluoride, it is almost completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than the Torpedo enzyme, suggesting that enhanced 'breathing motions' of the mouse enzyme relative to the Torpedo enzyme may explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in the Torpedo enzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approaches the active-site serine in both mouse and Torpedo acetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride is able to approach the active-site serine of mouse acetylcholinesterase, but remains trapped above the bottleneck in the Torpedo enzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:2.

The thioesterase domain of the fengycin biosynthesis cluster: a structural base for the macrocyclization of a non-ribosomal lipopeptide.

Many secondary metabolic peptides from bacteria and fungi are produced by non-ribosomal peptide synthetases (NRPS) where the final step of biosynthesis is often catalysed by designated thioesterase domains. Here, we report the 1.8A crystal structure of the fengycin thioesterase (FenTE) from Bacillus subtilis F29-3, which catalyses the regio- and stereoselective release and macrocyclization of the antibiotic fengycin from the NRPS template. A structure of the PMSF-inactivated FenTE domain suggests the location of the oxyanion hole and the binding site of the C-terminal residue l-Ile11 of the lipopeptide. Using a combination of docking, molecular dynamics simulations and in vitro activity assays, a model of the FenTE-fengycin complex was derived in which peptide cyclization requires strategic interactions with residues lining the active site canyon.

Crystal structure of carboxylesterase from Pseudomonas fluorescens, an alpha/beta hydrolase with broad substrate specificity.

BACKGROUND: A group of esterases, classified as carboxylesterases, hydrolyze carboxylic ester bonds with relatively broad substrate specificity and are useful for stereospecific synthesis and hydrolysis of esters. One such carboxylesterase from Pseudomonas fluorescens is a homodimeric enzyme, consisting of 218-residue subunits. It shows a limited sequence similarity to some members of the alpha/beta hydrolase superfamily. Although crystal structures of a number of serine esterases and lipases have been reported, structural information on carboxylesterases is very limited. This study was undertaken in order to provide such information and to understand a structural basis for the substrate specificity of this carboxylesterase. RESULTS: In this study, the crystal structure of carboxylesterase from P. fluorescens has been determined by the isomorphous replacement method and refined to 1.8 A resolution. Each subunit consists of a central seven-stranded beta sheet flanked by six alpha helices. The structure reveals the catalytic triad as Ser 114-His 199-Asp 168. The structure of the enzyme in complex with the inhibitor phenylmethylsulfonyl fluoride has also been determined and refined to 2.5 . The inhibitor is covalently attached to Ser 114 of both subunits, with the aromatic ring occupying a hydrophobic site defined by the aliphatic sidechains of Leu23, Ile58, Ile70, Met73 and Val170. No large structural changes are observed between the free and inhibitor-bound structures. CONCLUSIONS: Carboxylesterase from P. fluorescens has the alpha/beta hydrolase fold and the Ser-His-Asp catalytic triad. The active-site cleft in each subunit is formed by the six loops covering the catalytic serine residue. Three of the active-site loops in each subunit are involved in a head-to-head subunit interaction to form a dimer; it may be these extra structural elements, not seen in other esterases, that account for the inability of carboxylesterase to hydrolyze long chain fatty acids. As a result of dimerization, the active-site clefts from the two subunits merge to form holes in the dimer. The active-site clefts are relatively open and thus the catalytic residues are exposed to the solvent. An oxyanion hole, formed by nitrogen atoms of Leu23 and Gln115, is present in both the free and inhibitor-bound structures. An open active site, as well as a large binding pocket for the acid part of substrates, in P. fluorescens carboxylesterase may contribute to its relatively broad substrate specificity.