Manco G

General

Full name : Manco Giuseppe

First name : Giuseppe

Mail : Consiglio Nazionale delle Ricerche\; Istituto di Biochimica delle Proteine\; Via P. Castellino 111\; Naples\; 80131

Zip Code :

City :

Country : Italy

Email : g.manco@ibp.cnr.it

Phone : +39816132296

Fax :

Website :

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References (46)

Title : Development of a Qualitative Test to Detect the Presence of Organophosphate Pesticides on Fruits and Vegetables - De Luca_2023_Life.(Basel)_13_
Author(s) : De Luca V , Mandrich L , Manco G
Ref : Life (Basel) , 13 : , 2023
Abstract : BACKGROUND: In recent decades, the use of pesticides in agriculture has increased at a fast pace, highlighting safety problems for the environment and human health, which in turn has made it necessary to develop new detection and decontamination systems for pesticides. METHODS: A new qualitative test capable of detecting the presence of pesticides on fruits and vegetables by using thermostable enzymes was discovered, and the test was carried out on apples and aubergines. The contaminating pesticides were extracted from fruits with acetonitrile and analyzed with a biosensor system based on the thermostable esterase EST2 immobilized on a nitrocellulose filter. This enzyme is irreversibly inhibited mainly in the presence of organophosphates pesticides. Therefore, by observing esterase activity inhibition, we revealed the presence of residual pesticides on the fruits and vegetables. RESULTS: By analyzing the rate of esterase activity inhibition, we predicted that residual pesticides are present on the surface of the fruits. When we cleaned the fruits by washing them in the presence of the phosphotriesterase SsoPox before the detection of the esterase activity on filters, we observed a full recovery of the activity for apples and 30% for aubergines, indicating that the enzymatic decontamination of organophosphates pesticides took place. CONCLUSIONS: The reported method permitted us to assess the pesticides present on the vegetables and their decontamination.
ESTHER : De Luca_2023_Life.(Basel)_13_
PubMedSearch : De Luca_2023_Life.(Basel)_13_
PubMedID: 36836850

Title : Development of a Qualitative Test to Detect the Presence of Organophosphate Pesticides on Fruits and Vegetables - De Luca_2023_Life_13_490
Author(s) : De Luca V , Mandrich L , Manco G
Ref : Life , 13 :490 , 2023
Abstract : Background: In recent decades, the use of pesticides in agriculture has increased at a fast pace, highlighting safety problems for the environment and human health, which in turn has made it necessary to develop new detection and decontamination systems for pesticides. Methods: A new qualitative test capable of detecting the presence of pesticides on fruits and vegetables by using thermostable enzymes was discovered, and the test was carried out on apples and aubergines. The contaminating pesticides were extracted from fruits with acetonitrile and analyzed with a biosensor system based on the thermostable esterase EST2 immobilized on a nitrocellulose filter. This enzyme is irreversibly inhibited mainly in the presence of organophosphates pesticides. Therefore, by observing esterase activity inhibition, we revealed the presence of residual pesticides on the fruits and vegetables. Results: By analyzing the rate of esterase activity inhibition, we predicted that residual pesticides are present on the surface of the fruits. When we cleaned the fruits by washing them in the presence of the phosphotriesterase SsoPox before the detection of the esterase activity on filters, we observed a full recovery of the activity for apples and 30% for aubergines, indicating that the enzymatic decontamination of organophosphates pesticides took place. Conclusions: The reported method permitted us to assess the pesticides present on the vegetables and their decontamination.
ESTHER : De Luca_2023_Life_13_490
PubMedSearch : De Luca_2023_Life_13_490
PubMedID:
Gene_locus related to this paper: aliac-est2

Title : A mesophilic phosphotriesterase-like lactonase shows high stability and proficiency as quorum quenching enzyme - Marone_2023_Chem.Biol.Interact_14ChEPon_383_110657
Author(s) : Marone M , Porzio E , Lampitella EA , Manco G
Ref : Chemico-Biological Interactions , 383 :110657 , 2023
Abstract : The problem of biofilm formation is a serious concern under various pathological conditions such as extensive burns, wounds in diabetic patients, bedsores, cystic fibrosis, nosocomial infections from implantable medical devices such as catheters, valves, etc. Environmental diffusion of biofilm (in pools, wet floors, industrial food plants) that could represent a reservoir of antibiotic resistant bacteria constitues an additional issue. In this work is described a lactonase from Rhodococcus erythropolis, a phosphotriesterase-like lactonase (PLL) enzyme, which has already been studied in the past and can be used for containment of biofilm formation. The protein is 28% and 40% identical with respect to the Pseudomonas diminuta PTE and the thermostable Saccharolobus solfataricus SsoPox respectively. The protein was obtained starting from a synthetic His-tagged gene, expressed in E. coli, purified and further characterized. New properties, not previously known or deducible from its sequence, have been highlighted. These properties are: the enzyme is thermophilic and thermostable even though it originates from a mesophilic bacterium; the enzyme has a long (months) shelf life at 4 degreesC; the enzyme is not only stable to low concentrations of the oxidant H(2)O(2) but even activated by it at high concentrations; the enzyme proved to be a proficient quorum quenching enzyme, able to hydrolase acyl-homoserine lactones 3oxoC12-HSL and C4-HSL, and can inhibit up to 60% the formation of Pseudomonas aeruginosa (PAO1) biofilm. These different properties make the lactonase useful to fight resistant bacteria that induce inflammatory and infectious processes mediated by the quorum sensing mechanism.
ESTHER : Marone_2023_Chem.Biol.Interact_14ChEPon_383_110657
PubMedSearch : Marone_2023_Chem.Biol.Interact_14ChEPon_383_110657
PubMedID: 37573927

Title : Use of biosensors for rapid and sensitive detection of pesticides in food samples for food safety chemical risk assessment - Garefalaki_2022_EFSA.J_20_e200922
Author(s) : Garefalaki V , Manco G , Porzio E
Ref : EFSA J , 20 :e200922 , 2022
Abstract : The utility of pesticides in the agricultural field is unquestionable, but at the same time pesticide use presents serious hazards to the environment and the human health. For that reason, detection of pesticides and their biotransformation products in food is of utmost importance. According to previous studies, esterase-based biosensors have been proposed as a viable and efficient solution for the detection of organophosphate pesticides. In this project, a double mutant of the thermostable esterase-2 (EST2) from Alicyclobacillus acidocaldarius was studied as a potential biosensor, for its ability to detect residual amounts of pesticides. Initial characterisation of the enzyme was performed, that included determination of optimal pH, thermophilicity, as well as kinetic analysis. Subsequently, the enzyme was studied by enzymatic activity assays with and without the presence of various organophosphate compounds. The effect of the organophosphates on the enzymatic activity was measured and complete inhibition of the enzyme was observed after incubation with paraoxon. These experiments were followed by an additional method involving labelling of the enzyme with a fluorescent probe. In this case, the effect of different pesticides on the EST2 enzyme was monitored by measuring the fluorescence quenching upon addition to the enzyme. Fourteen compounds were screened with this method and significant fluorescence quenching was observed in the presence of paraoxon and methyl-paraoxon when used in equimolar amounts with the enzyme in the range of nanomolar. This biosensor has been also used to test the presence of pesticides in real food samples, like fruits and juices. This research represents a starting point to develop effective fluorescence-based biosensors aiming at the screening of mutants with different pesticide selectivity profiles. The use of this enzyme-based biosensor can have applications in the field of food traceability as well as environmental monitoring, to control the presence of toxic chemicals, in particular organophosphate pesticides.
ESTHER : Garefalaki_2022_EFSA.J_20_e200922
PubMedSearch : Garefalaki_2022_EFSA.J_20_e200922
PubMedID: 36531285

Title : High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers - Restaino_2018_BMC.Biotechnol_18_18
Author(s) : Restaino OF , Borzacchiello MG , Scognamiglio I , Fedele L , Alfano A , Porzio E , Manco G , De Rosa M , Schiraldi C
Ref : BMC Biotechnol , 18 :18 , 2018
Abstract : BACKGROUND: Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process. RESULTS: Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U.L(- 1) and 47.1 U.L(- 1).h(- 1) for SacPox and to 8700 U.L(- 1) and 180.6 U.L(- 1).h(- 1) for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 +/- 2.0%, suitable for industrial applications. CONCLUSIONS: In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process.
ESTHER : Restaino_2018_BMC.Biotechnol_18_18
PubMedSearch : Restaino_2018_BMC.Biotechnol_18_18
PubMedID: 29558934

Title : Enzymatic detoxication: a sustainable means of degrading toxic organophosphate pesticides and chemical warfare nerve agents - Manco_2018_J.Chem.Technol.Biotechnol_93_2064
Author(s) : Manco G , Porzio E , Suzumoto Y
Ref : J Chem Technol Biotechnol , 93 :2064 , 2018
Abstract : Organophosphorus (OP) compounds are highly toxic molecules mainly used as pesticides. OP compounds also include nerve gases used in the past as chemical warfare agents and collectively OP pesticides and nerve gases are referred to as nerve agents (NA). An intensive, widespread use of pesticides since the 20th century has resulted in the emergence of an urgent global issue concerning both environment and human health. In addition, past terroristic acts and the recent dramatic events in Syria highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and detoxification of these compounds in stored bulks, on critical surfaces and media (food, water and air) and for in vivo prophylaxes and therapies.OP compounds, act as covalent inhibitors of acetylcholinesterase (AChE) in the nerve system of vertebrates, thus posing a substantial threat to the ecosystem. In order to address a strong demand for the establishment of an environmental monitoring system and remediation process for NA, an increasing number of studies have been focused on the enzymatic degradation in vitro. Use of enzymes for detoxication and decontamination of toxic NA could provide a long-term benefit as it is environmentally friendly compared with conventional methods such as chemical treatments and incineration. This review presents an overview of the current state of enzymatic detoxification research against NA. This includes the detailed characterization and protein engineering for the improvement in NA-degrading activities of such enzymes. Research on biosensors for NA detection and identification, although important in the field, has not been treated in this review. Instead special attention has been paid to the Phosphotriesterase-Like-Lactonase (PLL) enzyme family. Several PLL enzymes have been isolated from hyperthermophilic Archaea or thermophilic/extremophilic Bacteria, and exhibit exceptional thermal stability. Extremophilic PLLs therefore hold promise for potential industrial application towards NA detoxification.
ESTHER : Manco_2018_J.Chem.Technol.Biotechnol_93_2064
PubMedSearch : Manco_2018_J.Chem.Technol.Biotechnol_93_2064
PubMedID:

Title : Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents - Porzio_2018_Sci.Rep_8_13773
Author(s) : Porzio E , Bettazzi F , Mandrich L , Del Giudice I , Restaino OF , Laschi S , Febbraio F , De Luca V , Borzacchiello MG , Carusone TM , Worek F , Pisanti A , Porcaro P , Schiraldi C , De Rosa M , Palchetti I , Manco G
Ref : Sci Rep , 8 :13773 , 2018
Abstract : Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.
ESTHER : Porzio_2018_Sci.Rep_8_13773
PubMedSearch : Porzio_2018_Sci.Rep_8_13773
PubMedID: 30214052

Title : Direct detection of organophosphate compounds in water by a fluorescence-based biosensing device - Carullo_2018_Sens.Actuators.B.Chem_255_3257
Author(s) : Carullo P , Chino M , Cetrangolo GP , Terreri S , Lombardi A , Manco G , Cimmino A , Febbraio F
Ref : Sensors and Actuators B: Chemical , 255 :3257 , 2018
Abstract : The main drawbacks in the use of acetylcholinesterase-based biosensors are their susceptibility to inhibition by too many chemicals, their limited time-stability, and the constant need for a supply of substrates for the measurements. In order to offset these deficiencies, we have addressed our studies towards the thermophilic esterase 2 from A. acidocaldarious, which shows a high specificity and affinity towards organophosphates and a high resistance under raw operative conditions. In particular, we have investigated the possibility of measuring the binding of organophosphates to the protein by using a fluorescent probe covalently linked near the active site. We have produced a mutant where the serine 35, a residue located at the entrance of the alcohol binding site, has been replaced by a cysteine residue. The addition of 1,5-IAEDANS as a fluorescent probe to the thiol group of the mutant-protein did not affect the capability of the enzyme to bind the paraoxon and its stability or instability over time. We have set up a continuous flow system based on a re-circulating solution of the probe-enzyme complex through a fluorimetric flow cell inside a spectrofluorimeter. The addition of paraoxon aliquots has been detected in real-time by measuring the fluorescence quenching of the probe-enzyme complex. The fluorescence signals, as well as the enzyme activity, were not affected by dilution and organic solvent addition. These results support the development of biosensing devices for the continuous monitoring of organophosphate compounds.
ESTHER : Carullo_2018_Sens.Actuators.B.Chem_255_3257
PubMedSearch : Carullo_2018_Sens.Actuators.B.Chem_255_3257
PubMedID:
Gene_locus related to this paper: aliac-est2

Title : An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination - Del Giudice_2016_Biotechnol.Bioeng_113_724
Author(s) : Del Giudice I , Coppolecchia R , Merone L , Porzio E , Carusone TM , Mandrich L , Worek F , Manco G
Ref : Biotechnol Bioeng , 113 :724 , 2016
Abstract : In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1) . The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces. Biotechnol. Bioeng. 2016;113: 724-734. (c) 2015 Wiley Periodicals, Inc.
ESTHER : Del Giudice_2016_Biotechnol.Bioeng_113_724
PubMedSearch : Del Giudice_2016_Biotechnol.Bioeng_113_724
PubMedID: 26416557

Title : Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius - Pennacchio_2015_Extremophiles_19_1001
Author(s) : Pennacchio A , Mandrich L , Manco G , Trincone A
Ref : Extremophiles , 19 :1001 , 2015
Abstract : The enzymatic regioselective hydrolysis of (a) acetylated mono- to tetrasaccharides of different nature, (b) of acetylated aryl glycosides and (c) of different acetylated nucleosides was studied enlarging the portfolio of substrates that can be employed by the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. The reactions were optimised to the extent that the amount of enzyme needed was lowered of two orders of magnitude with respect to the previously reported reactions, namely from 4000 to 40 U of enzyme per reaction. New additional solvents were screened and dramatic changes in regioselectivity were observed depending on the amount and type of solvent used. For example, in the presence of 10 % DMF, only two alpha-D-glucose products 6-OH and 4,6-OH (in a 76:24 ratio) were detected, whereas with 25 % DMF, at least four products of similar amount were observed. This versatility adds specific value to the biocatalyst making possible the design of biocatalytic reactions with different hydrophobic ester substrates. As an additional remarkable example, EST2 catalysed with a good yield and high regioselectivity the hydrolysis of p-nitrophenyl beta-D-xylopyranoside triacetate producing only the monoacetylated derivative with acetyl group in 3-O-position, in 2 min. The results with nucleosides as substrates are particularly interesting. The peracetates of 3',5'-di-O-acetylthymidine are converted almost quantitatively (95 %) to the monoacetylated derivative possessing free secondary OH; this regioselectivity is complementary to hydrolysis/alcoholysis reactions catalysed by CAL-B lipase or to other microbial hydrolytic biocatalysts, generally giving products with free primary OH groups. A docking analysis was undertaken with all analysed substrates suggesting a structural interpretation of the results. In most of cases, the best pose of the selected substrate was in line with the observed regioselectivity.
ESTHER : Pennacchio_2015_Extremophiles_19_1001
PubMedSearch : Pennacchio_2015_Extremophiles_19_1001
PubMedID: 26216109
Gene_locus related to this paper: aliac-est2

Title : Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates - Tang_2014_Enzyme.Microb.Technol_55_107
Author(s) : Tang X , Liang B , Yi T , Manco G , Ilariapalchetti , Liu A
Ref : Enzyme Microb Technol , 55 :107 , 2014
Abstract : Organophosphates (OPs) widely exist in ecosystem as toxic substances, for which sensitive and rapid analytical methods are highly requested. In the present work, by using N-terminal of ice nucleation protein (INP) as anchoring motif, a genetically engineered Escherichia coli (E. coli) strain surface displayed mutant organophosphorus hydrolase (OPH) (S5) with improved enzyme activity was successfully constructed. The surface location of INP-OPH fusion was confirmed by SDS-PAGE analysis and enzyme activity assays. The OPH-displayed bacteria facilitate the hydrolysis of p-nitrophenol (PNP) substituted organophosphates to generate PNP, which can be detected spectrometrically at 410nm. Over 90% of the recombinant protein present on the surface of microbes demonstrated enhanced enzyme activity and long-term stability. The OPH activity of whole cells was 2.16U/OD600 using paraoxon as its substrate, which is the highest value reported so far. The optimal temperature for OPH activity was around 55 degrees C, and suspended cultures retained almost 100% of its activity over a period of one month at room temperature, exhibiting the better stability than free OPH. The recombinant E. coli strain could be employed as a whole-cell biocatalyst for detecting PNP substituted OPs at wider ranges and lower detection limits. Specifically, the linear ranges of the calibration curves were 0.5-150muM paraoxon, 1-200muM parathion and 2.5-200muM methyl parathion, and limits of detection were 0.2muM, 0.4muM and 1muM for paraoxon, parathion and methyl parathion, respectively (S/N=3). These results indicate that the engineered OPH strain is a promising multifunctional bacterium that could be used for further large-scale industrial and environmental applications.
ESTHER : Tang_2014_Enzyme.Microb.Technol_55_107
PubMedSearch : Tang_2014_Enzyme.Microb.Technol_55_107
PubMedID: 24411452

Title : Comprehensive analysis of surface charged residues involved in thermal stability in Alicyclobacillus acidocaldarius esterase 2 - Pezzullo_2013_Protein.Eng.Des.Sel_26_47
Author(s) : Pezzullo M , Del Vecchio P , Mandrich L , Nucci R , Rossi M , Manco G
Ref : Protein Engineering Des Sel , 26 :47 , 2013
Abstract : Here we report a comprehensive analysis through alanine-scanning mutagenesis of the contribution of surface ion pairs to the thermal stability of Alicyclobacillus acidocaldarius esterase 2 (EST2). We produced 16 single mutants, 4 double mutants corresponding to selected ion pairs R31/E118, E43/K102, R58/D130, D145/R148, 2 double mutants (R63A/R98A and E50A/D94A) involving residues of a large ion network on the protein surface and the double-mutant R98A/R148A meant to disrupt the R98 interactions within the said network and, contextually, the interaction between R148 and D145. The double-mutant E43A/E273K was obtained by chance. All selected residues were replaced with alanine except E91, which was mutated to a glycine and K102, which was changed to a glutamine. All 24 proteins were over-expressed in Escherichia coli, purified and characterized with respect to the main features. Structural stability data were compared with an in silico prediction of DeltaDeltaG values. Our study of the individual factors involved in thermostability and their structural interpretation reveals that the great stability of this thermophilic protein can be explained by the contribution of a few residues at the protein surface.
ESTHER : Pezzullo_2013_Protein.Eng.Des.Sel_26_47
PubMedSearch : Pezzullo_2013_Protein.Eng.Des.Sel_26_47
PubMedID: 23035254
Gene_locus related to this paper: aliac-est2

Title : A Further Biochemical Characterization of DrPLL the Thermophilic Lactonase from Deinococcus radiodurans - Mandrich_2013_Protein.Pept.Lett_20_36
Author(s) : Mandrich L , Di Gennaro S , Palma A , Manco G
Ref : Protein Pept Lett , 20 :36 , 2013
Abstract : Recently, the cloning of the ORF Dr0930 from Deinococcus radiodurans displaying, as primary activity, a lactonase activity and a promiscuous phosphotriesterase activity was reported. The crystal structure of the resulting recombinant enzyme has been solved, and many mutants have been generated in order to increase the phosphotriesterase activity, with the aim to reach the level of activity of the related pPTE from Pseudomonas diminuta. In this paper we report an additional biochemical characterization of DrPLL and show that this enzyme has an optimal temperature for catalysis of 85 C and possesses promiscuous carboxylesterase, phophodiesterase and thioesterase activities which were not previously described. A metal analysis was performed on the purified protein by inductively coupled plasma mass spectrometry (ICP-MS ELAN DRC-e), which confirmed the presence of Ni2+ as a main metal in the recombinant protein. Surprisingly, the specificity constants (s=kcat/KM) for the pNP-decanoate and pNP-dodecanoate esters were only one order of magnitude lower than that for the lactone substrate thio-buthyl-gamma-butyric-lactone (TBBL), and the KM value for TBBL was more than ten-fold higher than those for the esters. We named this enzyme DrPLL, based on its structural and biochemical features it belongs to the Phosphotriesterase Like Lactonase group, a small protein family belonging to the amidohydrolase superfamily.
ESTHER : Mandrich_2013_Protein.Pept.Lett_20_36
PubMedSearch : Mandrich_2013_Protein.Pept.Lett_20_36
PubMedID: 22789107

Title : Mn(2+) modulates the kinetic properties of an archaeal member of the PLL family - Porzio_2013_Chem.Biol.Interact_203_251
Author(s) : Porzio E , Di Gennaro S , Palma A , Manco G
Ref : Chemico-Biological Interactions , 203 :251 , 2013
Abstract : Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus acidocaldarius lactonase, showing low but significant and extremely thermostable paraoxonase activity. This enzyme, that we have named SacPox, is a member of the new described family of phosphotriesterase-like lactonases (PLLs). In this family the binuclear metal centre, which is involved in the catalytic machinery, has been poorly studied up to now. In this work we describe the expression of the protein in presence of different metals showing Mn(2+) to support the higher activity. The enzyme has been over-expressed, purified and characterized as a Mn(2+)-containing enzyme by inductive plasma coupled mass spectrometry (ICP-MS), showing also surprising kinetic differences in comparison with the cadmium-containing enzyme. The Mn(2+) containing enzyme was about 30-fold more efficient with paraoxon as substrate and more stable than the Cd(2+) counterpart, even though the Mn(2+) affinity for the binuclear metal centre is apparently lower. These results increase our knowledge of the biochemical characteristics of SacPox mainly with regard to the metal-ions modulation of function.
ESTHER : Porzio_2013_Chem.Biol.Interact_203_251
PubMedSearch : Porzio_2013_Chem.Biol.Interact_203_251
PubMedID: 23174457

Title : Enzyme Promiscuity in the Hormone-Sensitive Lipase Family of Proteins - Manco_2012_Protein.Pept.Lett_19_144
Author(s) : Manco G , Merone L , Porzio E , Feng Y , Mandrich L
Ref : Protein Pept Lett , 19 :144 , 2012
Abstract : The number of enzymes endowed with the capacity to catalyse other reactions than the main, physiological one, a feature that has been called promiscuity, is increasing at a fast pace. Promiscuity is a highly pervasive phenomenon that is present at each level of life complexity. For enzymes, promiscuity encompasses interesting aspects related to their physiological role, evolution and biotechnological applications. Herein, at first we will describe some general aspects of enzyme promiscuity and then we will report some examples from the alpha/beta hydrolase superfamily of proteins, with particular emphasis to the hormone-sensitive lipase family.
ESTHER : Manco_2012_Protein.Pept.Lett_19_144
PubMedSearch : Manco_2012_Protein.Pept.Lett_19_144
PubMedID: 21933124
Gene_locus related to this paper: human-LIPE

Title : Non-lipolytic and lipolytic sequence-related carboxylesterases: a comparative study of the structure-function relationships of rabbit liver esterase 1 and bovine pancreatic bile-salt-activated lipase - Chahinian_2010_Biochim.Biophys.Acta_1801_1195
Author(s) : Chahinian H , Fantini J , Garmy N , Manco G , Sarda L
Ref : Biochimica & Biophysica Acta , 1801 :1195 , 2010
Abstract : To differentiate esterases from lipases at the structure-function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116-123 in bBAL is deleted in rLE. This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase-lipid adsorption complex at interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site.
ESTHER : Chahinian_2010_Biochim.Biophys.Acta_1801_1195
PubMedSearch : Chahinian_2010_Biochim.Biophys.Acta_1801_1195
PubMedID: 20655391

Title : Hyperthermophilic phosphotriesterases\/lactonases for the environment and human health - Mandrich_2010_Environ.Technol_31_1115
Author(s) : Mandrich L , Merone L , Manco G
Ref : Environ Technol , 31 :1115 , 2010
Abstract : In the last decades the idea to use enzymes for environmental bioremediation has been more and more proposed and, in the light of this, new solutions have been suggested and detailed studies on some classes of enzymes have been performed. In particular, our attention in the last few years has been focused on the enzymes belonging to the amidohydrolase superfamily. Several members of this superfamily are endowed with promiscuous activities. The term 'catalytic promiscuity' describes the capability of an enzyme to catalyse different chemical reactions, called secondary activities, at the active site responsible for the main activity. Recently, a new family of microbial lactonases with promiscuous phosphotriesterase activity, dubbed PTE-Like Lactonase (PLL), has been ascribed to the amidohydrolase superfamily. Among members of this family are enzymes found in the archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, which show high thermophilicity and thermal resistance. Enzymes showing phosphotriesterase activity are attractive from a biotechnological point of view because they are capable of hydrolysing the organophosphate phosphotriesters (OPs), a class of synthetic compounds employed worldwide both as insecticides and chemical warfare agents. Furthermore, from a basic point of view, studies of catalytic promiscuity offer clues to understand natural evolution of enzymes and to translate this into in vitro adaptation of enzymes to specific human needs. Thermostable enzymes able to hydrolyse OPs are considered good candidates for the set-up of efficient detoxification tools.
ESTHER : Mandrich_2010_Environ.Technol_31_1115
PubMedSearch : Mandrich_2010_Environ.Technol_31_1115
PubMedID: 20718294

Title : Improving the promiscuous nerve agent hydrolase activity of a thermostable archaeal lactonase - Merone_2010_Bioresour.Technol_101_9204
Author(s) : Merone L , Mandrich L , Porzio E , Rossi M , Muller S , Reiter G , Worek F , Manco G
Ref : Bioresour Technol , 101 :9204 , 2010
Abstract : The thermostable Phosphotriesterase-Like Lactonase from Sulfolobus solfataricus (SsoPox) hydrolyzes lactones and, at a lower rate, neurotoxic organophosphorus compounds. The persistent demand of detoxification tools in the field of agricultural wastes and restoring of conditions after terrorist acts prompted us to exploit SsoPox as a "starter" to evolve its ancillary nerve agents hydrolytic capability. A directed evolution strategy yielded, among several variants, the single mutant W263F with k(cat) and specificity constant against paraoxon 16- and 6-fold enhanced, respectively, compared to the wild type. Furthermore, a phenomenon of enzyme activation by SDS has been observed, which allowed to increase those values 150- and 28-fold, respectively. The activity of SsoPox against the deadly nerve gas Cyclosarin has been reported for the first time and proved to be substantially unaffected for variant W263F. Finally, outperforming efficiency of W263F was demonstrated, under severe stressing conditions, with respect to the best known phosphotriesterase PTE from Brevundimonas diminuta.
ESTHER : Merone_2010_Bioresour.Technol_101_9204
PubMedSearch : Merone_2010_Bioresour.Technol_101_9204
PubMedID: 20667718

Title : Structural and kinetic overview of the carboxylesterase EST2 from alicyclobacillus acidocaldarius: a comparison with the other members of the HSL family - Mandrich_2009_Protein.Pept.Lett_16_1189
Author(s) : Mandrich L , Merone L , Manco G
Ref : Protein Pept Lett , 16 :1189 , 2009
Abstract : Thermophilic and hyperthermophilic carboxylesterases (EC 3.1.1.1) are excellent model systems for studying structure function relationships as well as in vitro and in vivo evolution and possible biotechnological applications. In this paper we review the main aspect of one of most studied microbial representative of the hormone sensitive lipase family (HSL), namely carboxylesterase 2 (EST2) from Alicyclobacillus acidocaldarius.
ESTHER : Mandrich_2009_Protein.Pept.Lett_16_1189
PubMedSearch : Mandrich_2009_Protein.Pept.Lett_16_1189
PubMedID: 19508183
Gene_locus related to this paper: aliac-est2

Title : Use of esterase activities for the detection of chemical neurotoxic agents - Manco_2009_Protein.Pept.Lett_16_1225
Author(s) : Manco G , Nucci R , Febbraio F
Ref : Protein Pept Lett , 16 :1225 , 2009
Abstract : The quest for a quick and easy detection of the neurotoxin levels in the environment has fostered the search for systems alternative to currently employed analytical methods such as spectrophotometer, gas-liquid chromatography, thin-layer chromatography, and more recently mass spectrometry. These drawbacks lead to intense research efforts to develop biosensor devices for the determination of these compounds. In this review, we present an overview of the actual development of research in neurotoxin detection by using enzymatic biosensors based on esterase activity, in particular cholinesterases, and carboxylesterases. Detection by enzymatic activity could be carried out measuring the hydrolysis products or the residual enzymatic activity after inhibition, using a transducer system that makes possible the correlation between the determined activity and the analyte concentration. Several transducer systems were adopted for the neurotoxins identification using esterases, including electrochemical, optical, conductimetric and piezoelectric procedures. The differences in the used transducer determine the final sensitivity and specificity of the biosensor. Moreover, a brief description of immobilization procedure, that is an important step in the biosensor development and could affect the final characteristic of biosensor (sensibility, stability, response time and reproducibility), was accomplished. Final considerations on advantages and problems, related to actual development of these technologies, and its prospective were discussed.
ESTHER : Manco_2009_Protein.Pept.Lett_16_1225
PubMedSearch : Manco_2009_Protein.Pept.Lett_16_1225
PubMedID: 19508179

Title : Evolution in the amidohydrolase superfamily: substrate-assisted gain of function in the E183K mutant of a phosphotriesterase-like metal-carboxylesterase - Mandrich_2009_Biochemistry_48_5602
Author(s) : Mandrich L , Manco G
Ref : Biochemistry , 48 :5602 , 2009
Abstract : The recent specialization for utilization of pesticides reported for Pseudomonas diminuta phosphotriesterase (pPTE) strongly suggests that this activity evolved from an enzyme endowed with promiscuous phosphotriesterase activity. Such a putative "generalist" enzyme was recently proposed to be a member of the new phoshotriesterase-like lactonase family (PLL). The promiscuous carboxylesterase and phosphodiesterase activities detected in pPTE and PLLs in turn paved the way for the prediction of the existence in nature of PTE-like enzymes with predominant carboxylesterase or phosphodiesterase activities. An "in silico" analysis of the related Mesorhizobium loti ORF MLL7664 and the biochemical characterization demonstrated its prominent carboxylesterase and low phosphotriesterase specificity. On the basis of sequence similarity with the phosphotriesterase homology protein from Escherichia coli and the carboxylesterase activity, we called it phosphotriesterase-like carboxylesterase (MloPLC). The carboxylesterase activity is strictly dependent on divalent cations, and as such MloPLC is the first phosphotriesterase-like metal-carboxylesterase characterized to date. In related enzymes of the amidohydrolase superfamily either glutamate or carboxylated lysine substitutes for MloPLC glutamate 183 and the residue appear invariantly involved in maintaining the structural integrity of the binuclear metal center. Accordingly, we changed Glu-183 to lysine or glutamine. All the tested activities were completely abolished in the E183Q mutant, while only a residual phosphotriesterase activity could be detected in the E183K mutant. Surprisingly, in the latter mutant a parallel 650-fold specificity increase in bis-p-nitrophenyl-phosphate (BpNP-P) was observed, turning MloPLC from a carboxylesterase into a phosphodiesterase. Chemical, structural, and kinetic data strongly suggested that K183 is not carboxylated and that the gain of the new function is assisted by the substrate.
ESTHER : Mandrich_2009_Biochemistry_48_5602
PubMedSearch : Mandrich_2009_Biochemistry_48_5602
PubMedID: 19438255

Title : Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius - Mandrich_2008_Proteins_71_1721
Author(s) : Mandrich L , Menchise V , Alterio V , De Simone G , Pedone C , Rossi M , Manco G
Ref : Proteins , 71 :1721 , 2008
Abstract : Recent mutagenic and molecular modelling studies suggested a role for glycine 84 in the putative oxyanion loop of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. A 114 times decrease of the esterase catalytic activity of the G84S mutant was observed, without changes in the thermal stability. The recently solved three-dimensional (3D) structure of EST2 in complex with a HEPES molecule permitted to demonstrate that G84 (together with G83 and A156) is involved in the stabilization of the oxyanion through a hydrogen bond from its main chain NH group. The structural data in this case did not allowed us to rationalize the effect of the mutation, since this hydrogen bond was predicted to be unaltered in the mutant. Since the mutation could shed light on the role of the oxyanion loop in the HSL family, experiments to elucidate at the mechanistic level the reasons of the observed drop in k (cat) were devised. In this work, the kinetic and structural features of the G84S mutant were investigated in more detail. The optimal temperature and pH for the activity of the mutated enzyme were found significantly changed (T = 65 degrees C and pH = 5.75). The catalytic constants K (M) and V(max) were found considerably altered in the mutant, with ninefold increased K (M) and 14-fold decreased V(max), at pH 5.75. At pH 7.1, the decrease in k (cat) was much more dramatic. The measurement of kinetic constants for some steps of the reaction mechanism and the resolution of the mutant 3D structure provided evidences that the observed effects were partly due to the steric hindrance of the S84-OH group towards the ester substrate and partly to its interference with the nucleophilic attack of a water molecule on the second tetrahedral intermediate.
ESTHER : Mandrich_2008_Proteins_71_1721
PubMedSearch : Mandrich_2008_Proteins_71_1721
PubMedID: 18076040
Gene_locus related to this paper: aliac-est2

Title : Irreversible inhibition of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius - Febbraio_2008_Extremophiles_12_719
Author(s) : Febbraio F , D'Andrea SE , Mandrich L , Merone L , Rossi M , Nucci R , Manco G
Ref : Extremophiles , 12 :719 , 2008
Abstract : Kinetic studies of irreversible inhibition in recent years have received growing attention owing to their relevance to problems of basic scientific interest as well as to their practical importance. Our studies have been devoted to the characterization of the effects that well-known acetylcholinesterase irreversible inhibitors exert on a carboxylesterase (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius. In particular, sulfonyl inhibitors and the organophosphorous insecticide diethyl-p-nitrophenyl phosphate (paraoxon) have been studied. The incubation of EST2 with sulfonyl inhibitors resulted in a time-dependent inactivation according to a pseudo-first-order kinetics. On the other hand, the EST2 inactivation process elicited by paraoxon, being the inhibition reaction completed immediately after the inhibitor addition, cannot be described as a pseudo-first-order kinetics but is better considered as a high affinity inhibition. The values of apparent rate constants for paraoxon inactivation were determined by monitoring the enzyme/substrate reaction in the presence of the inhibitor, and were compared with those of the sulfonyl inhibitors. The protective effect afforded by a competitive inhibitor on the EST2 irreversible inhibition, and the reactivation of a complex enzyme/irreversible-inhibitor by hydroxylamine and 2-PAM, were also investigated. The data have been discussed in the light of the recently described dual substrate binding mode of EST2, considering that the irreversible inhibitors employed were able to discriminate between the two different binding sites.
ESTHER : Febbraio_2008_Extremophiles_12_719
PubMedSearch : Febbraio_2008_Extremophiles_12_719
PubMedID: 18622571
Gene_locus related to this paper: aliac-est2

Title : Structural basis for natural lactonase and promiscuous phosphotriesterase activities - Elias_2008_J.Mol.Biol_379_1017
Author(s) : Elias M , Dupuy J , Merone L , Mandrich L , Porzio E , Moniot S , Rochu D , Lecomte C , Rossi M , Masson P , Manco G , Chabriere E
Ref : Journal of Molecular Biology , 379 :1017 , 2008
Abstract : Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 A resolution) and in complex with a quorum-sensing lactone mimic at 2.0 A resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.
ESTHER : Elias_2008_J.Mol.Biol_379_1017
PubMedSearch : Elias_2008_J.Mol.Biol_379_1017
PubMedID: 18486146

Title : SSoNDelta and SSoNDeltalong: two thermostable esterases from the same ORF in the archaeon Sulfolobus solfataricus? - Mandrich_2007_Archaea_2_109
Author(s) : Mandrich L , Pezzullo M , Rossi M , Manco G
Ref : Archaea , 2 :109 , 2007
Abstract : Previously, we reported from the Sulfolobus solfataricus open reading frame (ORF) SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL) family and apparently having a deletion at the N-terminus, which we named SSoNDelta. Searching the recently reported Sulfolobus acidocaldarius genome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105) sharing high sequence similarity (82%) with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, named SSoNDeltalong, was 15-fold more active with the substrate p-nitrophenyl hexanoate than SSoNDelta. Furthermore, SSoNDeltalong and SSoNDelta displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship between S. solfataricus and S. acidocaldarius suggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression of SSoNDeltalong under specific metabolic conditions could be hypothesized.
ESTHER : Mandrich_2007_Archaea_2_109
PubMedSearch : Mandrich_2007_Archaea_2_109
PubMedID: 17350931

Title : A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus - Porzio_2007_Biochimie_89_625
Author(s) : Porzio E , Merone L , Mandrich L , Rossi M , Manco G
Ref : Biochimie , 89 :625 , 2007
Abstract : The phosphotriesterase PTE, identified in the soil bacterium Pseudomonas diminuta, is thought to have evolved in the last several decades to degrade the pesticide paraoxon with proficiency approaching the limit of substrate diffusion (k(cat)/K(M) of 4 x 10(7)M(-1)s(-1)). It belongs to the amidohydrolase superfamily, but its evolutionary origin remains obscure. The enzyme has important potentiality in the field of the organophosphate decontamination. Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus solfataricus, showing low but significant and extremely thermostable paraoxonase activity (k(cat)/K(M) of 4 x 10(3)M(-1)s(-1)). Looking for other thermostable phosphotriesterases we assayed, among others, crude extracts of Sulfolobus acidocaldarius and detected activity. Since the genome of S. acidocaldarius has been recently reported, we identified there an open reading frame highly related to the S. solfataricus enzyme. The gene was cloned, the protein overexpressed in Escherichia coli, purified, and proven to have paraoxonase activity. A comparative analysis detected some significant differences between the two archaeal enzymes.
ESTHER : Porzio_2007_Biochimie_89_625
PubMedSearch : Porzio_2007_Biochimie_89_625
PubMedID: 17337320

Title : The latent promiscuity of newly identified microbial lactonases is linked to a recently diverged phosphotriesterase - Afriat_2006_Biochemistry_45_13677
Author(s) : Afriat L , Roodveldt C , Manco G , Tawfik DS
Ref : Biochemistry , 45 :13677 , 2006
Abstract : In essence, evolutionary processes occur gradually, while maintaining fitness throughout. Along this line, it has been proposed that the ability of a progenitor to promiscuously catalyze a low level of the evolving activity could facilitate the divergence of a new function by providing an immediate selective advantage. To directly establish a role for promiscuity in the divergence of natural enzymes, we attempted to trace the origins of a bacterial phosphotriesterase (PTE), an enzyme thought to have evolved for the purpose of degradation of a synthetic insecticide introduced in the 20th century. We surmised that PTE's promiscuous lactonase activity may be a vestige of its progenitor and tested homologues annotated as "putative PTEs" for lactonase and phosphotriesterase activity. We identified three genes that define a new group of microbial lactonases dubbed PTE-like lactonases (PLLs). These enzymes proficiently hydrolyze various lactones, and in particular quorum-sensing N-acyl homoserine lactones (AHLs), and exhibit much lower promiscuous phosphotriesterase activities. PLLs share key sequence and active site features with PTE and differ primarily by an insertion in one surface loop. Given their biochemical and biological function, PLLs are likely to have existed for many millions of years. PTE could have therefore evolved from a member of the PLL family while utilizing its latent promiscuous paraoxonase activity as an essential starting point.
ESTHER : Afriat_2006_Biochemistry_45_13677
PubMedSearch : Afriat_2006_Biochemistry_45_13677
PubMedID: 17105187

Title : Use of an inhibitor to identify members of the hormone-sensitive lipase family - Ben Ali_2006_Biochemistry_45_14183
Author(s) : Ben Ali Y , Chahinian H , Petry S , Muller G , Lebrun R , Verger R , Carriere F , Mandrich L , Rossi M , Manco G , Sarda L , Abousalham A
Ref : Biochemistry , 45 :14183 , 2006
Abstract : Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.
ESTHER : Ben Ali_2006_Biochemistry_45_14183
PubMedSearch : Ben Ali_2006_Biochemistry_45_14183
PubMedID: 17115713
Gene_locus related to this paper: human-LIPE

Title : Alicyclobacillus acidocaldarius thermophilic esterase EST2's activity in milk and cheese models - Mandrich_2006_Appl.Environ.Microbiol_72_3191
Author(s) : Mandrich L , Manco G , Rossi M , Floris E , Jansen-van den Bosch T , Smit G , Wouters JA
Ref : Applied Environmental Microbiology , 72 :3191 , 2006
Abstract : The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30 degrees C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls.
ESTHER : Mandrich_2006_Appl.Environ.Microbiol_72_3191
PubMedSearch : Mandrich_2006_Appl.Environ.Microbiol_72_3191
PubMedID: 16672457
Gene_locus related to this paper: aliac-est2

Title : Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family - Mandrich_2005_J.Mol.Biol_345_501
Author(s) : Mandrich L , Merone L , Pezzullo M , Cipolla L , Nicotra F , Rossi M , Manco G
Ref : Journal of Molecular Biology , 345 :501 , 2005
Abstract : A superposition between the structures of Alicyclobacillus acidocaldarius esterase 2 (EST2) and Burkholderia cepacia lipase, the latter complexed with a phosphonate inhibitor, allowed us to hypothesize for the EST2 N terminus a role in restricting the access to the active site and therefore in modulating substrate specificity. In order to test this hypothesis we generated by site-directed mutagenesis some truncated versions of EST2 and its double mutant M211S/R215L (S/L) at the N terminus. In parallel, an analysis of the Sulfolobus solfataricus P2 genome allowed us to identify a gene coding for a putative esterase of the HSL family having a natural deletion of the corresponding region. The product of this gene and the above-mentioned EST2 mutants were expressed in Escherichia coli, purified and characterised. These studies support the notion that the N terminus affects substrate specificity other than several other enzyme parameters. Although the deletions afforded a tenfold and 550-fold decrease in catalytic efficiency towards the best substrate pNP-hexanoate at 50 degrees C for EST2 and S/L, respectively, the analysis of the specific activities with different triacylglycerols with respect to pNP-hexanoate showed that their ratios were higher for deleted versus non-deleted enzymes, on all tested substrates. In particular, the above ratios for glyceryl tridecanoate were 30-fold and 14-fold higher in S/L and EST2 deleted forms, respectively, compared with their full-length versions. This behaviour was confirmed by the analysis of the S.solfataricus esterase, which showed similar specific activities on pNP-hexanoate and triacylglycerols; in addition, higher activities on the latter substrates were observed in comparison with EST2, S/L and their deleted forms. Finally, a dramatic effect on thermophilicity and thermostability in the EST2 deleted forms was observed. This is the first report highlighting the importance of the "cap" domain in the HSL family, since the N terminus partly contributes to the building up of this structure.
ESTHER : Mandrich_2005_J.Mol.Biol_345_501
PubMedSearch : Mandrich_2005_J.Mol.Biol_345_501
PubMedID: 15581894
Gene_locus related to this paper: burce-lipaa

Title : Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases - Chahinian_2005_Biochim.Biophys.Acta_1738_29
Author(s) : Chahinian H , Ben Ali Y , Abousalham A , Petry S , Mandrich L , Manco G , Canaan S , Sarda L
Ref : Biochimica & Biophysica Acta , 1738 :29 , 2005
Abstract : We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.
ESTHER : Chahinian_2005_Biochim.Biophys.Acta_1738_29
PubMedSearch : Chahinian_2005_Biochim.Biophys.Acta_1738_29
PubMedID: 16325466

Title : A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis - De Simone_2004_J.Biol.Chem_279_6815
Author(s) : De Simone G , Mandrich L , Menchise V , Giordano V , Febbraio F , Rossi M , Pedone C , Manco G
Ref : Journal of Biological Chemistry , 279 :6815 , 2004
Abstract : The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites.
ESTHER : De Simone_2004_J.Biol.Chem_279_6815
PubMedSearch : De Simone_2004_J.Biol.Chem_279_6815
PubMedID: 14617621
Gene_locus related to this paper: aliac-est2

Title : The crystal structure of an EST2 mutant unveils structural insights on the H group of the carboxylesterase\/lipase family - De Simone_2004_J.Mol.Biol_343_137
Author(s) : De Simone G , Menchise V , Alterio V , Mandrich L , Rossi M , Manco G , Pedone C
Ref : Journal of Molecular Biology , 343 :137 , 2004
Abstract : Esterase 2 (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius is a thermostable serine hydrolase belonging to the H group of the esterase/lipase family. This enzyme hydrolyzes monoacylesters of different acyl-chain length and various compounds with industrial interest. EST2 displays an optimal temperature at 70 degrees C and maximal activity with pNP-esters having acyl-chain bearing from six to eight carbon atoms. EST2 mutants with different substrate specificity were also designed, generated by site-directed mutagenesis, and biochemically characterized. To better define at structural level the enzyme reaction mechanism, a crystallographic analysis of one of these mutants, namely M211S/R215L, was undertaken. Here we report its three-dimensional structure at 2.10A resolution. Structural analysis of the enzyme revealed an unexpected dimer formation as a consequence of a domain-swapping event involving its N-terminal region. This phenomenon was absent in the case of the enzyme bound to an irreversible inhibitor having optimal substrate structural features. A detailed comparison of the enzyme structures before and following binding to this molecule showed a movement of the N-terminal helices resulting from a trans-cis isomerization of the F37-P38 peptide bond. These findings suggest that this carboxylesterase presents two distinct structural arrangements reminiscent of the open and closed forms already reported for lipases. Potential biological implications associated with the observed quaternary reorganization are here discussed in light of the biochemical properties of other lipolytic members of the H group.
ESTHER : De Simone_2004_J.Mol.Biol_343_137
PubMedSearch : De Simone_2004_J.Mol.Biol_343_137
PubMedID: 15381425
Gene_locus related to this paper: aliac-est2

Title : Analysis of thermal adaptation in the HSL enzyme family - Mandrich_2004_J.Mol.Biol_335_357
Author(s) : Mandrich L , Pezzullo M , Del Vecchio P , Barone G , Rossi M , Manco G
Ref : Journal of Molecular Biology , 335 :357 , 2004
Abstract : The recently solved three-dimensional (3D) structures of two thermostable members of the carboxylesterase/lipase HSL family, namely the Alicyclobacillus (formerly Bacillus) acidocaldarius and Archaeoglobus fulgidus carboxylesterases (EST2 and AFEST, respectively) were compared with that of the mesophilic homologous counterpart Brefeldine A esterase from Bacillus subtilis. Since the 3D homology models of other members of the HSL family were also available, we performed a structural alignment with all these sequences. The resulting alignment was used to assess the amino acid "traffic rule" in the HSL family. Quite surprisingly, the data were in very good agreement with those recently reported from two independent groups and based on the comparison of a huge number of homologous sequences from the genus Bacillus, Methanococcus and Deinococcus/Thermus. Taken as a whole, the data point to the statistical meaning of defined amino acid conversions going from psychrophilic to hyperthermophilic sequences. We identified and mapped several such changes onto the EST2 structure and observed that such mutations were localized mostly in loops regions or alpha-helices and were mostly excluded from the active site. A site-directed mutagenesis of two of the identified residues confirmed they were involved in thermal stability.
ESTHER : Mandrich_2004_J.Mol.Biol_335_357
PubMedSearch : Mandrich_2004_J.Mol.Biol_335_357
PubMedID: 14659763

Title : Crystallization and preliminary X-ray diffraction studies of Aes acetyl-esterase from Escherichia coli - Sorrentino_2003_Acta.Crystallogr.D.Biol.Crystallogr_59_1846
Author(s) : Sorrentino N , De Simone G , Menchise V , Mandrich L , Rossi M , Manco G , Pedone C
Ref : Acta Crystallographica D Biol Crystallogr , 59 :1846 , 2003
Abstract : The acetyl-esterase Aes from Escherichia coli, which belongs to the HSL group of the esterase/lipase superfamily, has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 8000 as a precipitant and magnesium chloride as an additive. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 110.0, b = 190.6, c = 218.6 A. A complete data set has been collected to 2.5 A resolution at the Elettra synchrotron source, Trieste using a single frozen crystal. Packing density considerations agree with 10-16 monomers in the asymmetric unit, with a corresponding solvent content of 61-38%.
ESTHER : Sorrentino_2003_Acta.Crystallogr.D.Biol.Crystallogr_59_1846
PubMedSearch : Sorrentino_2003_Acta.Crystallogr.D.Biol.Crystallogr_59_1846
PubMedID: 14501134
Gene_locus related to this paper: ecoli-Aes

Title : Effect of trifluoroethanol on the conformational stability of a hyperthermophilic esterase: a CD study - Del Vecchio_2003_Biophys.Chem_104_407
Author(s) : Del Vecchio P , Graziano G , Granata V , Barone G , Mandrich L , Rossi M , Manco G
Ref : Biophysical Chemistry , 104 :407 , 2003
Abstract : The conformational stability of the hyperthermophilic esterase AFEST from Archeoglobus fulgidus against the denaturing action of 2,2,2-trifluoroethanol (TFE) has been investigated by means of circular dichroism (CD) measurements. At room temperature far-UV and near-UV CD spectra point out the occurrence of a co-operative transition from the native structure to a denatured state characterized by a high content of alpha-helix. The TFE concentration at half-completion of the transition proves to be 3.5 M (25% v v(-1)), by recording the molar ellipticity at both 222 and 276 nm. Thermal transition curves of AFEST in the absence and in the presence of TFE indicate a significant stability decrease on increasing the TFE concentration. The denaturation temperature is 99 degrees C for native AFEST, but becomes 85 degrees C at 1.4 M TFE (10% v v(-1)), and 56 degrees C at 2.8 M TFE (20% v v(-1)). It is also shown that, even though AFEST is very resistant to temperature, its resistance towards the denaturing action of TFE is similar to that of mesophilic proteins, including an esterase from Escherichia coli, AES. The proposal of a general mechanism for the TFE action on globular proteins leads to a reliable rationale of experimental data.
ESTHER : Del Vecchio_2003_Biophys.Chem_104_407
PubMedSearch : Del Vecchio_2003_Biophys.Chem_104_407
PubMedID: 12878309
Gene_locus related to this paper: arcfu-estea

Title : The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex. Implications for the regulation of carbohydrate metabolism - Mandrich_2002_J.Biol.Chem_277_48241
Author(s) : Mandrich L , Caputo E , Martin BM , Rossi M , Manco G
Ref : Journal of Biological Chemistry , 277 :48241 , 2002
Abstract : Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the alpha-galactosidase from E. coli. The E. coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because alpha-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli.
ESTHER : Mandrich_2002_J.Biol.Chem_277_48241
PubMedSearch : Mandrich_2002_J.Biol.Chem_277_48241
PubMedID: 12374803
Gene_locus related to this paper: ecoli-Aes

Title : The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus - De Simone_2001_J.Mol.Biol_314_507
Author(s) : De Simone G , Menchise V , Manco G , Mandrich L , Sorrentino N , Lang D , Rossi M , Pedone C
Ref : Journal of Molecular Biology , 314 :507 , 2001
Abstract : The crystal structure of AFEST, a novel hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus, complexed with a sulphonyl derivative, has been determined and refined to 2.2 A resolution. This enzyme, which has recently been classified as a member of the hormone- sensitive-lipase (H) group of the esterase/lipase superfamily, presents a canonical alpha/beta hydrolase core, shielded on the C-terminal side by a cap region composed of five alpha-helices. It contains the catalytic triad Ser160, His285 and Asp255, whereby the nucleophile is covalently modified and the oxyanion hole formed by Gly88, Gly89 and Ala161. A structural comparison of AFEST with its mesophilic and thermophilic homologues, Brefeldin A esterase from Bacillus subtilis (BFAE) and EST2 from Alicyclobacillus acidocaldarius, reveals an increase in the number of intramolecular ion pairs and secondary structure content, as well as a significant reduction in loop extensions and ratio of hydrophobic to charged surface area. The variety of structural differences suggests possible strategies for thermostabilization of lipases and esterases with potential industrial applications.
ESTHER : De Simone_2001_J.Mol.Biol_314_507
PubMedSearch : De Simone_2001_J.Mol.Biol_314_507
PubMedID: 11846563
Gene_locus related to this paper: arcfu-estea

Title : Residues at the active site of the esterase 2 from Alicyclobacillus acidocaldarius involved in substrate specificity and catalytic activity at high temperature - Manco_2001_J.Biol.Chem_276_37482
Author(s) : Manco G , Mandrich L , Rossi M
Ref : Journal of Biological Chemistry , 276 :37482 , 2001
Abstract : The recently solved three-dimensional structure of the thermophilic esterase 2 from Alicyclobacillus acidocaldarius allowed us to have a snapshot of an enzyme-sulfonate complex, which mimics the second stage of the catalytic reaction, namely the covalent acyl-enzyme intermediate. The aim of this work was to design, by structure-aided analysis and to generate by site-directed and saturation mutagenesis, EST2 variants with changed substrate specificity in the direction of preference for monoacylesters whose acyl-chain length is greater than eight carbon atoms. Positions 211 and 215 of the polypeptide chain were chosen to introduce mutations. Among five variants with single and double amino acid substitutions, three were obtained, M211S, R215L, and M211S/R215L, that changed the catalytic efficiency profile in the desired direction. Kinetic characterization of mutants and wild type showed that this change was achieved by an increase in k(cat) and a decrease in K(m) values with respect to the parental enzyme. The M211S/R215L specificity constant for p-nitrophenyl decanoate substrate was 6-fold higher than the wild type. However, variants M211T, M211S, and M211V showed strikingly increased activity as well as maximal activity with monoacylesters with four carbon atoms in the acyl chain, compared with the wild type. In the case of mutant M211T, the k(cat) for p-nitrophenyl butanoate was 2.4-fold higher. Overall, depending on the variant and on the substrate, we observed improved catalytic activity at 70 degrees C with respect to the wild type, which was a somewhat unexpected result for an enzyme with already high k(cat) values at high temperature. In addition, variants with altered specificity toward the acyl-chain length were obtained. The results were interpreted in the context of the EST2 three-dimensional structure and a proposed catalytic mechanism in which k(cat), e.g. the limiting step of the reaction, was dependent on the acyl chain length of the ester substrate.
ESTHER : Manco_2001_J.Biol.Chem_276_37482
PubMedSearch : Manco_2001_J.Biol.Chem_276_37482
PubMedID: 11447219
Gene_locus related to this paper: aliac-est2

Title : Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus - Manco_2000_Protein.Eng_13_197
Author(s) : Manco G , Camardella L , Febbraio F , Adamo G , Carratore V , Rossi M
Ref : Protein Engineering , 13 :197 , 2000
Abstract : The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.
ESTHER : Manco_2000_Protein.Eng_13_197
PubMedSearch : Manco_2000_Protein.Eng_13_197
PubMedID: 10775661
Gene_locus related to this paper: arcfu-AF1763

Title : Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus - Manco_2000_Arch.Biochem.Biophys_373_182
Author(s) : Manco G , Giosue E , D'Auria S , Herman P , Carrea G , Rossi M
Ref : Archives of Biochemistry & Biophysics , 373 :182 , 2000
Abstract : A new esterase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus, reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family, was cloned by means of the polymerase chain reaction from the A. fulgidus genome. In order to compare the biochemical properties of this putative hyperthermophilic enzyme with those of the homologous, thermophilic member of HSL group, namely Alicyclobacillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble and active form at 20 mg/liter of E. coli culture, was purified to homogeneity and characterized. The enzyme, a 35.5-kDa monomeric protein, was demonstrated to be a B"-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-hexanoate with K(m) and k(cat) values of 11 +/- 3 microM (mean +/- SD, n = 3) and 1014 +/- 38 s(-1) (mean +/- SD, n = 3), respectively, at 70 degrees C and pH 7.1. Inactivation by diethylpyrocarbonate, phenylmethylsulfonylfluoride, diisopropylfosfofluoridate (DFP), and physostigmine, as well as labeling with [(3)H]DFP, supported our previous suggestion of a catalytic triad made up of Ser(160)-His(285)-Asp(255). The sequence identity with the thermostable A. acidocaldarius EST2 was 42.5%. The enzyme proved to be much more stable than its Alicyclobacillus counterpart. The conformational dynamics of the two proteins were investigated by frequency-domain fluorometry and anisotropy decay and the activity/stability/temperature relationship was discussed.
ESTHER : Manco_2000_Arch.Biochem.Biophys_373_182
PubMedSearch : Manco_2000_Arch.Biochem.Biophys_373_182
PubMedID: 10620337

Title : A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase - De Simone_2000_J.Mol.Biol_303_761
Author(s) : De Simone G , Galdiero S , Manco G , Lang D , Rossi M , Pedone C
Ref : Journal of Molecular Biology , 303 :761 , 2000
Abstract : EST2 is a novel thermophilic carboxylesterase, isolated and cloned from Alicyclobacillus (formerly Bacillus) acidocaldarius, which optimally hydrolyses esters with acyl chain lengths of six to eight carbon atoms at 70 degrees C. On the basis of the amino acid sequence homology, it has been classified as a member of the mammalian hormone-sensitive lipase (HSL) subfamily. The crystal structure of EST2, complexed with a sulphonyl derivative, has been determined at 2.6 A resolution by a multiple wavelength anomalous diffraction experiment on a seleno-methionine derivative. EST2 presents a canonical alpha/beta hydrolase core, shielded at the C-terminal side by a cap region built up of five helices. It contains the lipase-like catalytic triad, Ser155, His282 and Asp252, whereby the nucleophile is covalently modified. This allows an unambiguous view of the putative active site of EST2, detecting the oxyanion hole, in whose formation the amino acid sequence motif His81-Gly82-Gly83-Gly84 is involved, and the hydrophobic binding pocket for the acyl chain. The structural model here reported provides the first example of a transition state analogue of an esterase/lipase belonging to the HSL group, thus affording useful information for the design of medical inhibitors. Moreover, as the first X-ray structure of a thermophilic carboxylesterase, the comparison with its mesophilic homologue, the Brefeldin A esterase (BFAE) from Bacillus subtilis, allows the identification of putative determinants of thermal stability.
ESTHER : De Simone_2000_J.Mol.Biol_303_761
PubMedSearch : De Simone_2000_J.Mol.Biol_303_761
PubMedID: 11061974
Gene_locus related to this paper: aliac-est2

Title : The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus - D'Auria_2000_Proteins_40_473
Author(s) : D'Auria S , Herman P , Lakowicz JR , Tanfani F , Bertoli E , Manco G , Rossi M
Ref : Proteins , 40 :473 , 2000
Abstract : The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.
ESTHER : D'Auria_2000_Proteins_40_473
PubMedSearch : D'Auria_2000_Proteins_40_473
PubMedID: 10861939
Gene_locus related to this paper: aliac-est2

Title : Crystallization and preliminary X-ray diffraction studies of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius - De Simone_1999_Acta.Crystallogr.D.Biol.Crystallogr_55_1348
Author(s) : De Simone G , Manco G , Galdiero S , Lombardi A , Rossi M , Pavone V
Ref : Acta Crystallographica D Biol Crystallogr , 55 :1348 , 1999
Abstract : EST2, a thermophilic carboxylesterase from Alicyclobacillus acidocaldarius, belonging to the HSL group of the esterase/lipase superfamily, has been crystallized for the first time. Ammonium sulfate was used as a precipitant and the crystallization proceeded at pH 7.8. The crystals belong to space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 78.8, c = 106. 4 A. A complete data set has been collected at the synchrotron source Elettra in Trieste to 2.4 A resolution, using a single frozen crystal.
ESTHER : De Simone_1999_Acta.Crystallogr.D.Biol.Crystallogr_55_1348
PubMedSearch : De Simone_1999_Acta.Crystallogr.D.Biol.Crystallogr_55_1348
PubMedID: 10393304
Gene_locus related to this paper: aliac-est2

Title : Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2 - Manco_1999_Protein.Sci_8_1789
Author(s) : Manco G , Febbraio F , Adinolfi E , Rossi M
Ref : Protein Science , 8 :1789 , 1999
Abstract : The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure-driven multiple sequence alignment with remote homologous protein of known three-dimensional (3D) structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure-driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative "oxyanion hole" involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.
ESTHER : Manco_1999_Protein.Sci_8_1789
PubMedSearch : Manco_1999_Protein.Sci_8_1789
PubMedID: 10493580
Gene_locus related to this paper: aliac-est2

Title : Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily - Manco_1998_Biochem.J_332 ( Pt 1)_203
Author(s) : Manco G , Adinolfi E , Pisani FM , Ottolina G , Carrea G , Rossi M
Ref : Biochemical Journal , 332 ( Pt 1) :203 , 1998
Abstract : We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B'-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11+/-2 microM (mean+/-S.D., n=3) and 6610+/-880 s-1 (mean+/-S.D., n=3) respectively at 70 degreesC and pH7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (+/-)-3-bromo-5-(hydroxymethyl)-Delta2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relationship is discussed in relation to those of the homologous members of the HSL group.
ESTHER : Manco_1998_Biochem.J_332 ( Pt 1)_203
PubMedSearch : Manco_1998_Biochem.J_332 ( Pt 1)_203
PubMedID: 9576869
Gene_locus related to this paper: aliac-est2