Recombinant production and characterization of a metal ion-independent Lysophospholipase from a hyperthermophilic archaeon Pyrococcus abyssi DSM25543.

Genome sequence of Pyrococcus abyssi DSM25543 contains a coding sequence (PAB_RS01410) for α/ß hydrolase (WP_010867387.1). Structural analysis revealed the presence of a consensus motif GXSXG and a highly conserved catalytic triad in the amino acid sequence of α/ß hydrolase that were characteristic features of lysophospholipases. A putative lysophospholipase from P. abyssi with its potential applications in oil degumming and starch processing was heterologously produced in E. coli Rosetta (DE3) pLysS in soluble form followed by its purification and characterization. The recombinant enzyme was found to be active at temperature of 40-90 °C and pH 5.5-7.0. However, the enzyme exhibited its optimum activity at 65 °C and pH 6.5. None of the metal ions (Mn2+, Mg2+, Ni2+, Cu2+, Fe2+, Co2+, Zn2+ and Ca2+) being tested had stimulatory effect on lysophospholipase activity. Km and Vmax for hydrolysis of 4-nitrophenyl butyrate were calculated to be 1 ± 0.089 mM and 1637 ± 24.434 U/mg, respectively. It is the first report on the soluble production and characterization of recombinant lysophospholipase from P. abyssi which exhibits its lipolytic activity in the absence of divalent metal ions. Broad substrate specificity, activity and stability at elevated temperatures make recombinant lysophospholipase an ideal candidate for potential industrial applications.

Crystalline State Determines the Potency of Galectin-10 Protein Assembly to Induce Inflammation.

Protein crystallization is a prevalent phenomenon existing in the formation of intricate protein-assembled structures in living cells. Whether the crystallization of a protein would exert a specific biological function, however, remains poorly understood. Here, we reconstructed a recombinant galectin-10 (gal-10) protein and artificially engineered a gal-10 protein assembly in two distinguishable states: i.e., an insoluble crystalline state and a soluble state. The potency of the gal-10 protein in either the crystalline state or the soluble state to induce chemokine or cytokine release in the primary human nasal epithelial cells and nasal polyps derived from chronic rhinosinusitis patients with nasal polyps was investigated. The crystalline gal-10 upregulated the gene expression of chemokines or cytokines, including IL-1ß, IL-6, IL-8, TNF-α, and GM-CSF, in patient-derived primary cells and nasal polyps. In contrast, soluble gal-10 displayed a diminished potency to induce inflammation. Our results demonstrate that the gal-10 protein potency of activating inflammation is correlated with its crystalline state.

IPRO+/-: Computational Protein Design Tool Allowing for Insertions and Deletions.

Insertions and deletions (indels) in protein sequences alter the residue spacing along the polypeptide backbone and consequently open up possibilities for tuning protein function in a way that is inaccessible by amino acid substitution alone. We describe an optimization-based computational protein redesign approach centered around predicting beneficial combinations of indels along with substitutions and also obtain putative substrate-docked structures for these protein variants. This modified algorithmic capability would be of interest for enzyme engineering and broadly inform other protein design tasks. We highlight this capability by (1) identifying active variants of a bacterial thioesterase enzyme ('TesA) with experimental corroboration, (2) recapitulating existing active TEM-1 ß-Lactamase sequences of different sizes, and (3) identifying shorter 4-Coumarate:CoA ligases with enhanced in vitro activities toward non-native substrates. A separate PyRosetta-based open-source tool, Indel-Maker (http://www.maranasgroup.com/software.htm), has also been created to construct computational models of user-defined protein variants with specific indels and substitutions.

Discovery of small-molecule enzyme activators by activity-based protein profiling.

Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse-small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1-a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1's catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.

Characterisation of a lysophospholipase from Lactobacillus mucosae.

OBJECTIVE: In this study, we characterised a novel lysophospholipase (LysoPL) from the L. mucosae LM1 strain. The gene, LM-lysoPL, encoding LysoPL from L. mucosae LM1 was cloned, analyzed, and expressed. RESULTS: LM-lysoPL contained a conserved region and catalytic triad motif responsible for lysophospholipase activity. After purification, UHPLC-MS analysis showed that recombinant LM-LysoPL hydrolyzed phosphatidic acid, generating lysophosphatidic acid. The enzyme had greater hydrolytic activity against C16 and C18 fatty acids, indicating a preference for long-chain fatty acids. Enzymatic assays showed that the optimal pH and temperature of recombinant LM-LysoPL were 7 and 30 °C, respectively, and it was enzymatically active within a narrow pH range. CONCLUSIONS: To the best of our knowledge, this is the first study to identify and characterize a lysophospholipase from lactic acid bacteria. Our findings provide a basis for understanding the probiotic role of L. mucosae LM1 in the gut.

Structure-guided reshaping of the acyl binding pocket of 'TesA thioesterase enhances octanoic acid production in E. coli.

Medium-chain fatty acids (C6-C10) have attracted much attention recently for their unique properties compared to their long-chain counterparts, including low melting points and relatively higher carbon conversion yield. Thioesterase enzymes, which can catalyze the hydrolysis of acyl-ACP (acyl carrier protein) to release free fatty acids (FAs), regulate both overall FA yields and acyl chain length distributions in bacterial and yeast fermentation cultures. These enzymes typically prefer longer chain substrates. Herein, seeking to increase bacterial production of MCFAs, we conducted structure-guided mutational screening of multiple residues in the substrate-binding pocket of the E. coli thioesterase enzyme 'TesA. Confirming our hypothesis that enhancing substrate selectivity for medium-chain acyl substrates would promote overall MCFA production, we found that replacement of residues lining the bottom of the pocket with more hydrophobic residues strongly promoted the C8 substrate selectivity of 'TesA. Specifically, two rounds of saturation mutagenesis led to the identification of the 'TesARD-2 variant that exhibited a 133-fold increase in selectivity for the C8-ACP substrate as compared to C16-ACP substrate. Moreover, the recombinant expression of this variant in an E. coli strain with a blocked ß-oxidation pathway led to a 1030% increase in the in vivo octanoic acid (C8) production titer. When this strain was fermented in a 5-L fed-batch bioreactor, it produced 2.7 g/L of free C8 (45%, molar fraction) and 7.9 g/L of total free FAs, which is the highest-to-date free C8 titer to date reported using the E. coli type II fatty acid synthetic pathway. Thus, reshaping the substrate binding pocket of a bacterial thioesterase enzyme by manipulating the hydrophobicity of multiple residues altered the substrate selectivity and therefore fatty acid product distributions in cells. Our study demonstrates the relevance of this strategy for increasing titers of industrially attractive MCFAs as fermentation products.

The Charcot-Leyden crystal protein revisited-A lysopalmitoylphospholipase and more.

The Charcot-Leyden crystal protein (CLC-P), a constituent of human and not mouse eosinophils, is one of the most abundant proteins within human eosinophils. It has a propensity to form crystalline structures, Charcot-Leyden crystals, which are hallmarks in their distinctive extracellular crystalline forms as markers of eosinophilic inflammation. The functions of CLC-P within eosinophils have been uncertain. Although the action of CLC-P as a lysophospholipase has been questioned, assays of chromatographically purified CLC-P and crystal-derived CLC-P as well as studies of transfected recombinant CLC-P have consistently documented that CLC-P endogenously expresses lysophospholipase activity, releasing free palmitate from substrate lysopalmitoylphosphatidylcholine. Rather than acting solely as a hydrolytic enzyme to release palmitate from a lysolipid substrate, some other lysophospholipases function more dominantly as acyl-protein thioesterases (APTs), enzymes that catalyze the removal of thioester-linked, long chain fatty acids, such as palmitate, from cysteine residues of proteins. As such APTs participate in palmitoylation, a post-translational modification that can affect membrane localization, vesicular transport, and secretion. CLC-P has attributes of an APT. Thus, whereas CLC-P expresses inherent lysophospholipase activity, like some other lysophospholipase enzymes, it likely also functions in regulating the dynamic palmitoylation cycle, including, given its dominant subplasmalemmal location, at the human eosinophil's plasma membrane.

Hierarchical porous and hydrophilic metal-organic frameworks with enhanced enzyme activity.

Metal-organic frameworks (MOFs) for enzyme encapsulation-induced biomimetic mineralization under mild reaction conditions are commonly microporous and hydrophobic, which result in a rather high mass transfer resistance of the reactants and restrain the enzyme catalytic activity. Herein, we prepared a type of hierarchical porous and hydrophilic MOF through the biomimetic mineralization of enzymes, zinc ions, 2-methylimidazole, and lithocholic acid. The hierarchical porous structure accelerated the diffusion process of the reactants and the increased hydrophilicity conferred interfacial activity and increased the enzyme catalytic activity. The immobilized enzyme retained higher catalytic activity than the free enzyme and exhibited enhanced resistance to alkaline, organic, and high-temperature conditions. The nanobiocatalyst was reusable and showed long-term storage stability.

A Thermolabile Phospholipase B from Talaromyces marneffei GD-0079: Biochemical Characterization and Structure Dynamics Study.

Phospholipase B (EC 3.1.1.5) are a distinctive group of enzymes that catalyzes the hydrolysis of fatty acids esterified at the sn-1 and sn-2 positions forming free fatty acids and lysophospholipids. The structural information and catalytic mechanism of phospholipase B are still not clear. Herein, we reported a putative phospholipase B (TmPLB1) from Talaromyces marneffei GD-0079 synthesized by genome mining library. The gene (TmPlb1) was expressed and the TmPLB1 was purified using E. coli shuffle T7 expression system. The putative TmPLB1 was purified by affinity chromatography with a yield of 13.5%. The TmPLB1 showed optimum activity at 35 °C and pH 7.0. The TmPLB1 showed enzymatic activity using Lecithin (soybean > 98% pure), and the hydrolysis of TmPLB1 by 31P NMR showed phosphatidylcholine (PC) as a major phospholipid along with lyso-phospholipids (1-LPC and 2-LPC) and some minor phospholipids. The molecular modeling studies indicate that its active site pocket contains Ser125, Asp183 and His215 as the catalytic triad. The structure dynamics and simulations results explained the conformational changes associated with different environmental conditions. This is the first report on biochemical characterization and structure dynamics of TmPLB1 enzyme. The present study could be helpful to utilize TmPLB1 in food industry for the determination of food components containing phosphorus. Additionally, such enzyme could also be useful in Industry for the modifications of phospholipids.

Protein crystallization promotes type 2 immunity and is reversible by antibody treatment.

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

Thermodynamic insights into an interaction between ACYL-CoA-BINDING PROTEIN2 and LYSOPHOSPHOLIPASE2 in Arabidopsis.

Lysophospholipids (LPLs) are important lipid-signaling molecules in plants, of which lysophosphatidylcholine (lysoPC) is one of the most well-characterized LPLs, having important roles in plant stress responses. It is broken down by lysophospholipases, but the molecular mechanism involved in lysoPC degradation is unclear. Recombinant Arabidopsis thaliana ACYL-CoA-BINDING PROTEIN2 (AtACBP2) has been reported to bind lysoPC via its acyl-CoA-binding domain and also LYSOPHOSPHOLIPASE 2 (AtLYSOPL2) via its ankyrin repeats in vitro To investigate the interactions of AtACBP2 with AtLYSOPL2 and lysoPC in more detail, we conducted isothermal titration calorimetry with AtACBP270-354, an AtACBP2 derivative consisting of amino acids 70-354, containing both the acyl-CoA-binding domain and ankyrin repeats. We observed that the interactions of AtACBP270-354 with AtLYSOPL2 and lysoPC were both endothermic, favored by solvation entropy and opposed by enthalpy, with dissociation constants in the micromolar range. Of note, three AtLYSOPL2 catalytic triad mutant proteins (S147A, D268A, and H298A) bound lysoPC only weakly, with an exothermic burst and dissociation constants in the millimolar range. Furthermore, the binding affinity of lysoPC-premixed AtACBP270-354 to AtLYSOPL2 was 10-fold higher than that of AtACBP270-354 alone to AtLYSOPL2. We conclude that AtACBP2 may play a role in facilitating a direct interaction between AtLYSOPL2 and lysoPC. Our results suggest that AtACBP270-354 probably binds to lysoPC through a hydrophobic interface that enhances a hydrotropic interaction of AtACBP270-354 with AtLYSOPL2 and thereby facilitates AtLYSOPL2's lysophospholipase function.

A Brief History of Charcot-Leyden Crystal Protein/Galectin-10 Research.

Eosinophils are present in tissues, such as the respiratory tract, spleen, lymph nodes and blood vessels. The significant presence of eosinophils in these tissues are associated with various diseases, including asthma, allergies, acute myeloid leukemia, etc. Charcot-Leyden crystal protein/galectin-10 is overexpressed in eosinophils and has also been identified in basophils and macrophages. In human body, this protein could spontaneously form Charcot-Leyden crystal in lymphocytes or in the lysates of lymphocytes. At present, the role of Charcot-Leyden crystal protein/galectin-10 in lymphocytes is not fully understood. This review summarizes research progress on Charcot-Leyden crystal protein/galectin-10, with emphasis on its history, cellular distributions, relations to diseases, structures and ligand binding specificity.

Modeling and molecular dynamics indicate that snake venom phospholipase B-like enzymes are Ntn-hydrolases.

Phospholipase-B-like (SVPLB-like) enzymes are present in relatively small amounts in a number of venoms, however, their biological function and mechanisms of action are un-clear. A three-dimensional model of the SVPLB-like enzyme from Crotalus adamanteus was generated by homology modeling based on the crystal structures of bovine Ntn-hydrolyases and the modeled protein possesses conserved domains characteristic of Ntn-hydrolases. Molecular dynamics simulations indicate that activation by autocatalytic cleavage results in the removal of 25 amino acids which increases accessibility to the active site. SVPLB-like enzymes possess a highly reactive cysteine and are hence amidases that to belong to the N-terminal nucleophile (Ntn) hydrolase family. The Ntn-hydrolases (N-terminal nucleophile) form a superfamily of diverse enzymes that are activated autocatalytically; wherein the N-terminal catalytic nucleophile is implicated in the cleavage of the amide bond.

Characterization of a novel thiol activated phospholipase TAPLB1 from Trichosporon asahii MSR 54.

Phospholipases are hydrolytic enzymes that play crucial roles in vivo and also possess immense biotechnological potential. In the present study, the phospholipase B of Trichosporon asahii MSR54 was overexpressed in E. coli and characterized. The 68-kDa enzyme was monomeric in solution and possessed phospholipase, lysophospholipase, esterase and acyltransferase activities. It was maximally active at pH 8.0 and 40 °C. The enzyme retained >50% activity between pH 3.0-8.0 and had a half-life of 30 min at 60 °C. Its activity was not metal dependent and was stable in the presence of most metal ions. Its catalytic efficiency on lysophosphatidyl choline was 1.0 × 103 mM-1 h-1. Site directed mutagenesis revealed R121 (present in the GYRAMV motif), S194 (present in the conserved GLSGG motif) and D420 (present in LVDXGE motif) to be the crucial amino acid residues for esterolytic activity. S194 and D420 were also the catalytic amino acids for lysophospholipase and phospholipase activities of the enzymes, while R121 was not involved in catalysis of phospholipid substrates. Further, it was found that cysteine residues in C61 and C354 were involved in disulphide linkages that imparted the properties of thiol activation and thermostability, respectively.

Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy.

We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. Graphical Abstract ᅟ.

Molecular Basis of ABHD5 Lipolysis Activation.

Alpha-beta hydrolase domain-containing 5 (ABHD5), the defective gene in human Chanarin-Dorfman syndrome, is a highly conserved regulator of adipose triglyceride lipase (ATGL)-mediated lipolysis that plays important roles in metabolism, tumor progression, viral replication, and skin barrier formation. The structural determinants of ABHD5 lipolysis activation, however, are unknown. We performed comparative evolutionary analysis and structural modeling of ABHD5 and ABHD4, a functionally distinct paralog that diverged from ABHD5 ~500 million years ago, to identify determinants of ABHD5 lipolysis activation. Two highly conserved ABHD5 amino acids (R299 and G328) enabled ABHD4 (ABHD4 N303R/S332G) to activate ATGL in Cos7 cells, brown adipocytes, and artificial lipid droplets. The corresponding ABHD5 mutations (ABHD5 R299N and ABHD5 G328S) selectively disrupted lipolysis without affecting ATGL lipid droplet translocation or ABHD5 interactions with perilipin proteins and ABHD5 ligands, demonstrating that ABHD5 lipase activation could be dissociated from its other functions. Structural modeling placed ABHD5 R299/G328 and R303/G332 from gain-of-function ABHD4 in close proximity on the ABHD protein surface, indicating they form part of a novel functional surface required for lipase activation. These data demonstrate distinct ABHD5 functional properties and provide new insights into the functional evolution of ABHD family members and the structural basis of lipase regulation.

Recombinant expression, characterization, and application of a phospholipase B from Fusarium oxysporum.

In this study, a gene encoding a putative lipase from Fusarium oxysporum was optimized via codon optimization and expressed in Pichia pastoris KM71. The gene product was identified as a phospholipase B (PLB). The engineered P. pastoris was further cultured in a 3.6-L bioreactor. After optimization of the induction conditions, this system produced 6.6mgmL-1 protein and 6503.8UmL-1 PLB activity in the culture medium. Efficient expression of this PLB in P. pastoris should reduce the costs of production and application. The purified enzyme, with a specific activity of 1170Umg-1, was optimally active at pH 5.0 and 55°C. The results of a degumming experiment performed using the recombinant PLB showed that the phosphorus content of a test oil was decreased from 75.88ppm to 3.3ppm in 2h under optimal reaction conditions. This study provides a basis for the industrial use of F. oxysporum PLB in oil degumming applications.

Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy.

Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions. These methods have been used to verify the dimer stoichiometry of the Fluc F- ion channel and the dimerization equilibrium constant of the ClC-ec1 Cl-/H+ antiporter in lipid bilayers. This approach can be applied to any membrane protein system provided it can be purified, fluorescently labeled in a quantitative manner, and verified to be correctly folded by functional assays, even if the structure is not yet known.

Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism.

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.