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Nature Communications Jan 2017Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium...
Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium falciparum is highly variable depending on the commercial source. Here we identify a minor contaminant (pepstatin butyl ester) as the active anti-parasitic principle. We synthesize a series of derivatives and characterize an analogue (pepstatin hexyl ester) with low nanomolar activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. Recombinant PfPARE hydrolyses pepstatin esters and de-esterifies MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance.
Topics: Antimalarials; Drug Resistance; Esterases; Mutation; Pepstatins; Plasmodium falciparum; Prodrugs; Protozoan Proteins
PubMed: 28106035
DOI: 10.1038/ncomms14240 -
Microbial Cell Factories May 2017Pyrethroids are potentially harmful to human health and ecosystems. It is necessary to develop some efficient strategies to degrade pyrethroid residues. Biodegradation...
BACKGROUND
Pyrethroids are potentially harmful to human health and ecosystems. It is necessary to develop some efficient strategies to degrade pyrethroid residues. Biodegradation is generally considered as a safe, efficient, and inexpensive way to eliminate environmental contaminants. To date, although several pyrethroid-hydrolyzing esterases have been cloned, there has been no report about a pyrethroid hydrolase with high hydrolytic activity, good stability, and high productivity, indispensable enzymatic properties in practical biodegradation. Almost all pyrethroid hydrolases are intracellular enzymes, which require complex extraction protocols and present issues in terms of easy inactivation and low production.
RESULTS
In this study, random mutagenesis was performed on one pyrethroid-hydrolyzing esterase, Sys410, to enhance its activity and thermostability. Two beneficial mutations, A171V and D256N, were obtained by random mutagenesis and gave rise to the mutant M2. The mutant displayed ~1.5-fold improvement in the kcat/Km value and 2.46-fold higher catalytic activity. The optimal temperature was 10 °C higher than that of the wild-type enzyme (55 °C). The half-life at 40-65 °C was 3.3-310 times longer. It was surprising that M2 has a half-life of 12 h at 70 °C while Sys410 was completely inactivated at 70 °C. In addition, the desired gene was extracellularly expressed in a Pichia pastoris host system. The soluble expression level reached up to 689.7 mg/L. Remarkably, the enzyme could efficiently degrade various pyrethroids at moderate temperature for 15 min, exceeding a hydrolysis rate of 98%, which is the highest value ever reported.
CONCLUSIONS
This is the first report about random mutagenesis and secretory expression of pyrethroid-hydrolyzing esterase with high-level productivity and purity in P. pastoris. Broad substrate specificity, enhanced activity and thermostability make M2 an ideal candidate for the biodegradation of pyrethroid residues.
Topics: Biocatalysis; Biodegradation, Environmental; Cloning, Molecular; Directed Molecular Evolution; Enzyme Stability; Esterases; Gene Expression; Half-Life; Hydrogen-Ion Concentration; Hydrolases; Hydrolysis; Kinetics; Mutagenesis; Pyrethrins; Substrate Specificity; Temperature
PubMed: 28490329
DOI: 10.1186/s12934-017-0698-5 -
BMC Research Notes Sep 2019Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited,...
OBJECTIVE
Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost.
RESULTS
The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg L), and were secreted at moderate to high percentages of the total extracellular protein (51-94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U L) that could allow their direct application.
Topics: Batch Cell Culture Techniques; Esterases; Extracellular Space; Fermentation; Glucuronic Acid; Methanol; Pichia; Recombinant Proteins
PubMed: 31533815
DOI: 10.1186/s13104-019-4638-9 -
Molecules (Basel, Switzerland) Jul 2017The albumin molecule, in contrast to many other plasma proteins, is not covered with a carbohydrate moiety and can bind and transport various molecules of endogenous and...
The albumin molecule, in contrast to many other plasma proteins, is not covered with a carbohydrate moiety and can bind and transport various molecules of endogenous and exogenous origin. The enzymatic activity of albumin, the existence of which many scientists perceive skeptically, is much less studied. In toxicology, understanding the mechanistic interactions of organophosphates with albumin is a special problem, and its solution could help in the development of new types of antidotes. In the present work, the history of the issue is briefly examined, then our in silico data on the interaction of human serum albumin with soman, as well as comparative in silico data of human and bovine serum albumin activities in relation to paraoxon, are presented. Information is given on the substrate specificity of albumin and we consider the possibility of its affiliation to certain classes in the nomenclature of enzymes.
Topics: Animals; Cattle; Enzyme Activation; Esterases; Humans; Hydrolysis; Ligands; Models, Molecular; Molecular Conformation; Organophosphates; Protein Binding; Serum Albumin; Substrate Specificity
PubMed: 28718803
DOI: 10.3390/molecules22071201 -
International Journal of Molecular... Jan 2022Esterases and lipases can process amphiphilic esters used as drugs and prodrugs and impact their pharmacokinetics and biodistribution. These hydrolases can also process...
Esterases and lipases can process amphiphilic esters used as drugs and prodrugs and impact their pharmacokinetics and biodistribution. These hydrolases can also process ester components of drug delivery systems (DDSs), thus triggering DDSs destabilization with premature cargo release. In this study we tested and optimized assays that allowed us to quantify and compare individual esterase contributions to the degradation of substrates of increased lipophilicity and to establish limitations in terms of substrates that can be processed by a specific esterase/lipase. We have studied the impact of carbonic anhydrase; phospholipases A1, A2, C and D; lipoprotein lipase; and standard lipase on the hydrolysis of 4-nitrophenyl acetate, 4-nitrophenyl palmitate, DGGR and POPC liposomes, drawing structure-property relationships. We found that the enzymatic activity of these proteins was highly dependent on the lipophilicity of the substrate used to assess them, as expected. The activity observed for classical esterases was diminished when lipophilicity of the substrate increased, while activity observed for lipases generally increased, following the interfacial activation model, and was highly dependent on the type of lipase and its structure. The assays developed allowed us to determine the most sensitive methods for quantifying enzymatic activity against substrates of particular types and lipophilicity.
Topics: Carboxylic Ester Hydrolases; Cardiovascular System; Esterases; Esters; Hydrolysis; Kinetics; Lipase; Substrate Specificity; Tissue Distribution
PubMed: 35163184
DOI: 10.3390/ijms23031262 -
MicrobiologyOpen Oct 2018A novel esterase gene selected from metagenomic sequences of deep-sea hydrothermal vents was successfully expressed in Escherichia coli. The recombinant protein...
A novel esterase gene selected from metagenomic sequences of deep-sea hydrothermal vents was successfully expressed in Escherichia coli. The recombinant protein (est-OKK), which belongs to the lipolytic enzyme family V, exhibited high activity toward pNP-esters with short acyl chains and especially p-nitrophenyl butyrate. Site-mutagenesis results confirmed that est-OKK contains the nonclassical catalytic tetrad predicted by alignment and computational modeling. The est-OKK protein is a moderately thermophilic enzyme that is relatively thermostable, and highly salt-tolerant, which remained stable in 3 mol/L NaCl for 6 hr. The est-OKK protein showed the considerable alkalistability, displayed optimal activity at pH 9.0 and maintained approximately 70% of its residual activity after incubation at pH 10 for 4 hr. Furthermore, the est-OKK activity was strongly resistant to a variety of metal ions such as Co , Zn , Fe , Na , and K ; nonionic detergents such as Tween-20, Tween-80; and organic solvents such as acetone and isopropanol. Taken together, the novel esterase with unique characteristics may give us a new insight into the family V of lipolytic enzymes, and could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions or food and pharmaceutical industries.
Topics: Cloning, Molecular; DNA Mutational Analysis; Enzyme Stability; Escherichia coli; Esterases; Gene Expression; Hydrogen-Ion Concentration; Hydrothermal Vents; Metagenome; Pacific Ocean; Salts; Substrate Specificity; Temperature
PubMed: 29504251
DOI: 10.1002/mbo3.601 -
Clinical and Experimental Pharmacology... Jul 20101. High-density lipoprotein (HDL) is one of the major carriers of cholesterol in the blood. It attracts particular attention because, in contrast with other... (Review)
Review
1. High-density lipoprotein (HDL) is one of the major carriers of cholesterol in the blood. It attracts particular attention because, in contrast with other lipoproteins, many physiological functions of HDL influence the cardiovascular system in favourable ways unless HDL is modified pathologically. 2. The best known function of HDL is the capacity to promote cellular cholesterol efflux from peripheral cells and deliver cholesterol to the liver for excretion, thereby playing a key role in reverse cholesterol transport. The functions of HDL that have recently attracted attention include anti-inflammatory and anti-oxidant activities. High anti-oxidant and anti-inflammatory activities of HDL are associated with protection from cardiovascular disease. 3. Atheroprotective activities, as well as a functional deficiency of HDL, ultimately depend on the protein and lipid composition of HDL. Conversely, these activities are compromised in many pathological states associated with inflammation. 4. The focus of the present review is on the anti-oxidant and anti-inflammatory functions of HDL and its individual components in relation to protection from atherosclerosis.
Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Animals; Anti-Inflammatory Agents; Antioxidants; Aryldialkylphosphatase; Atherosclerosis; Cholesterol; Esterases; Glutathione Peroxidase; Humans; Lipoproteins, HDL; Mice; Oxidative Stress
PubMed: 20374263
DOI: 10.1111/j.1440-1681.2010.05380.x -
Acta Biomaterialia Apr 2018To identify and characterize specific esterases from S. mutans with degradative activity toward methacrylate-based resin monomers.
OBJECTIVES
To identify and characterize specific esterases from S. mutans with degradative activity toward methacrylate-based resin monomers.
METHODS
Out of several putative esterases, an esterase encoded in an Open Reading Frame as SMU_118c (The National Center for Biotechnology Information, NCBI), was found to have true hydrolase activities. SMU_118c was cloned, expressed, purified and further characterized for its respective hydrolytic activity towards ester-containing nitrophenyl substrates and the universal resin monomers bis-phenyl-glycidyl-dimethacrylate (bisGMA) and triethyleneglycol dimethacrylate (TEGDMA) at neutral (7.0) or cariogenic (5.5) pH. Mass spectrometry (MS) was used to verify the expression of SMU_118c protein in S. mutans UA159.
RESULTS
Similar to the whole cell activity of S. mutans, SMU_118c showed the highest affinity toward para-nitrophenyl acetate (pNPA) and para-nitrophenyl butyrate (pNPB) vs. ortho-nitrophenyl butyrate (oNPB) and butyrylthiocholine iodide (BTC) (p < 0.05). The esterase retained 60% of its activity after 21 days and hydrolyzed bisGMA at a higher rate than TEGDMA at both neutral and cariogenic pH (p < 0.001), similarly to the predominant human salivary esterase degradative activity. MS confirmed that SMU_118c is an intracellular protein in S. mutans UA159 and expressed under pathogenic (pH 5.5) growth conditions.
SIGNIFICANCE
The similarity in the activity profile to the whole S. mutans bacterial cell, the stability over time at cariogenic pH, the preference to hydrolyze bisGMA and confirmed expression profile suggest that SMU_118c could be a significant contributor to the whole bacterial degradative activity of S. mutans toward the degradation of resin composites, adhesives and the restoration-tooth interface, potentially accelerating restoration's failure.
STATEMENT OF SIGNIFICANCE
The current study builds upon our highly-cited previous study by Bourbia et al., (JDR, 2013) that reported on that the cariogenic bacterium, S. mutans has esterase-like activities that enable the bacterium to degrade dental composites and adhesives. The current submission is the first to report on the isolation and characterization of the specific esterase activity (SMU_118c) from S. mutans that is a significant contributor to the whole bacterial degradative activity toward the hydrolysis of dental resins. This activity compromises the restoration-tooth interface, increases interfacial bacterial microleakage (Kermanshahi et al., JDR 2010), potentially contributing to the pathogenesis of recurrent caries around resin composite restorations. This represent a significant contribution to the field of biomaterials and their clinical performance.
Topics: Bacterial Proteins; Dental Caries; Esterases; Humans; Hydrolysis; Recombinant Proteins; Resins, Synthetic; Streptococcus mutans
PubMed: 29496621
DOI: 10.1016/j.actbio.2018.02.020 -
Yakugaku Zasshi : Journal of the... 2015Esterases hydrolyze compounds containing ester, amide, and thioester bonds, causing prodrug activation or detoxification. Among esterases, carboxylesterases have been... (Review)
Review
Esterases hydrolyze compounds containing ester, amide, and thioester bonds, causing prodrug activation or detoxification. Among esterases, carboxylesterases have been studied in depth due to their ability to hydrolyze a variety of drugs. However, there are several drugs for which the involved esterase(s) is unknown. We found that flutamide, phenacetin, rifamycins (rifampicin, rifabutin, and rifapentine), and indiplon are hydrolyzed by arylacetamide deacetylase (AADAC), which is highly expressed in human liver and gastrointestinal tissues. Flutamide hydrolysis is considered associated with hepatotoxicity. Phenacetin, a prodrug of acetaminophen, was withdrawn due to side effects such as methemoglobinemia and renal failure. It was demonstrated in vitro and in vivo using mice that AADAC is responsible for phenacetin hydrolysis, which leads to methemoglobinemia. In addition, it was shown that AADAC-mediated hydrolysis attenuates the cytotoxicity of rifamycins. Thus AADAC plays critical roles in drug-induced toxicity. Another orphan esterase, α/β hydrolase domain containing 10 (ABHD10), was found responsible for deglucuronidation of acyl-glucuronides including mycophenolic acid acyl-glucuronide and probenecid acyl-glucuronide. Because acyl-glucuronides appear associated with toxicity, ABHD10 would function as a detoxification enzyme. The roles of orphan esterases are becoming increasingly understood. Further studies will facilitate our knowledge of the pharmacologic and toxicological significance of orphan esterases in drug therapy.
Topics: Animals; Carboxylic Ester Hydrolases; Drug-Related Side Effects and Adverse Reactions; Esterases; Humans; Hydrolases; Phenacetin; Rifamycins; Species Specificity
PubMed: 26521872
DOI: 10.1248/yakushi.15-00186 -
Applied and Environmental Microbiology Sep 2017Halotolerant enzymes are beneficial for industrial processes requiring high salt concentrations and low water activity. Most halophilic proteins are evolved to have...
Structural and Mechanistic Insights into the Improvement of the Halotolerance of a Marine Microbial Esterase by Increasing Intra- and Interdomain Hydrophobic Interactions.
Halotolerant enzymes are beneficial for industrial processes requiring high salt concentrations and low water activity. Most halophilic proteins are evolved to have reduced hydrophobic interactions on the surface and in the hydrophobic cores for their haloadaptation. However, in this study, we improved the halotolerance of a thermolabile esterase, E40, by increasing intraprotein hydrophobic interactions. E40 was quite unstable in buffers containing more than 0.3 M NaCl, and its and substrate affinity were both significantly reduced in 0.5 M NaCl. By introducing hydrophobic residues in loop 1 of the CAP domain and/or α7 of the catalytic domain in E40, we obtained several mutants with improved halotolerance, and the M3 S202W I203F mutant was the most halotolerant. ("M3" represents a mutation in loop 1 of the CAP domain in which residues R22-K23-T24 of E40 are replaced by residues Y22-K23-H24-L25-S26 of Est2.) Then we solved the crystal structures of the S202W I203F and M3 S202W I203F mutants to reveal the structural basis for their improved halotolerance. Structural analysis revealed that the introduction of hydrophobic residues W202 and F203 in α7 significantly improved E40 halotolerance by strengthening intradomain hydrophobic interactions of F203 with W202 and other residues in the catalytic domain. By further introducing hydrophobic residues in loop 1, the M3 S202W I203F mutant became more rigid and halotolerant due to the formation of additional interdomain hydrophobic interactions between the introduced Y22 in loop 1 and W204 in α7. These results indicate that increasing intraprotein hydrophobic interactions is also a way to improve the halotolerance of enzymes with industrial potential under high-salt conditions. Esterases and lipases for industrial application are often subjected to harsh conditions such as high salt concentrations, low water activity, and the presence of organic solvents. However, reports on halotolerant esterases and lipases are limited, and the underlying mechanism for their halotolerance is still unclear due to the lack of structures. In this study, we focused on the improvement of the halotolerance of a salt-sensitive esterase, E40, and the underlying mechanism. The halotolerance of E40 was significantly improved by introducing hydrophobic residues. Comparative structural analysis of E40 and its halotolerant mutants revealed that increased intraprotein hydrophobic interactions make these mutants more rigid and more stable than the wild type against high concentrations of salts. This study shows a new way to improve enzyme halotolerance, which is helpful for protein engineering of salt-sensitive enzymes.
Topics: Bacterial Proteins; Catalytic Domain; Enzyme Stability; Esterases; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Protein Structure, Tertiary; Seawater; Sodium Chloride
PubMed: 28733281
DOI: 10.1128/AEM.01286-17