Rossi M

References (39)

Title : Sustainable Drug Discovery of Multi-Target-Directed Ligands for Alzheimer's Disease - Rossi_2021_J.Med.Chem_64_4972
Author(s) : Rossi M , Freschi M , de Camargo Nascente L , Salerno A , de Melo Viana Teixeira S , Nachon F , Chantegreil F , Soukup O , Prchal L , Malaguti M , Bergamini C , Bartolini M , Angeloni C , Hrelia S , Soares Romeiro LA , Bolognesi ML
Ref : Journal of Medicinal Chemistry , 64 :4972 , 2021
Abstract : The multifactorial nature of Alzheimer's disease (AD) is a reason for the lack of effective drugs as well as a basis for the development of "multi-target-directed ligands" (MTDLs). As cases increase in developing countries, there is a need of new drugs that are not only effective but also accessible. With this motivation, we report the first sustainable MTDLs, derived from cashew nutshell liquid (CNSL), an inexpensive food waste with anti-inflammatory properties. We applied a framework combination of functionalized CNSL components and well-established acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) tacrine templates. MTDLs were selected based on hepatic, neuronal, and microglial cell toxicity. Enzymatic studies disclosed potent and selective AChE/BChE inhibitors (5, 6, and 12), with subnanomolar activities. The X-ray crystal structure of 5 complexed with BChE allowed rationalizing the observed activity (0.0352 nM). Investigation in BV-2 microglial cells revealed antineuroinflammatory and neuroprotective activities for 5 and 6 (already at 0.01 microM), confirming the design rationale.
ESTHER : Rossi_2021_J.Med.Chem_64_4972
PubMedSearch : Rossi_2021_J.Med.Chem_64_4972
PubMedID: 33829779
Gene_locus related to this paper: human-BCHE

Title : Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs - Bradley_2020_Nat.Chem.Biol_16_240
Author(s) : Bradley SJ , Molloy C , Valuskova P , Dwomoh L , Scarpa M , Rossi M , Finlayson L , Svensson KA , Chernet E , Barth VN , Gherbi K , Sykes DA , Wilson CA , Mistry R , Sexton PM , Christopoulos A , Mogg AJ , Rosethorne EM , Sakata S , John Challiss RA , Broad LM , Tobin AB
Ref : Nat Chemical Biology , 16 :240 , 2020
Abstract : Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including 'epileptic-like' seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD.
ESTHER : Bradley_2020_Nat.Chem.Biol_16_240
PubMedSearch : Bradley_2020_Nat.Chem.Biol_16_240
PubMedID: 32080630

Title : Molecular Hybridization as a Tool for Designing Multitarget Drug Candidates for Complex Diseases - Ivasiv_2019_Curr.Top.Med.Chem_19_1694
Author(s) : Ivasiv V , Albertini C , Goncalves AE , Rossi M , Bolognesi ML
Ref : Curr Top Med Chem , 19 :1694 , 2019
Abstract : Molecular hybridization is a well-exploited medicinal chemistry strategy that aims to combine two molecules (or parts of them) in a new, single chemical entity. Recently, it has been recognized as an effective approach to design ligands able to modulate multiple targets of interest. Hybrid compounds can be obtained by linking (presence of a linker) or framework integration (merging or fusing) strategies. Although very promising to combat the multifactorial nature of complex diseases, the development of molecular hybrids faces the critical issues of selecting the right target combination and the achievement of a balanced activity towards them, while maintaining drug-like-properties. In this review, we present recent case histories from our own research group that demonstrate why and how molecular hybridization can be carried out to address the challenges of multitarget drug discovery in two therapeutic areas that are Alzheimer's and parasitic diseases. Selected examples spanning from linker- to fragment- based hybrids will allow to discuss issues and consequences relevant to drug design.
ESTHER : Ivasiv_2019_Curr.Top.Med.Chem_19_1694
PubMedSearch : Ivasiv_2019_Curr.Top.Med.Chem_19_1694
PubMedID: 31237210

Title : Role of bifidobacteria in the hydrolysis of chlorogenic acid - Raimondi_2015_Microbiolopen_4_41
Author(s) : Raimondi S , Anighoro A , Quartieri A , Amaretti A , Tomas-Barberan FA , Rastelli G , Rossi M
Ref : Microbiologyopen , 4 :41 , 2015
Abstract : This study aimed to explore the capability of potentially probiotic bifidobacteria to hydrolyze chlorogenic acid into caffeic acid (CA), and to recognize the enzymes involved in this reaction. Bifidobacterium strains belonging to eight species occurring in the human gut were screened. The hydrolysis seemed peculiar of Bifidobacterium animalis, whereas the other species failed to release CA. Intracellular feruloyl esterase activity capable of hydrolyzing chlorogenic acid was detected only in B. animalis. In silico research among bifidobacteria esterases identified Balat_0669 as the cytosolic enzyme likely responsible of CA release in B. animalis. Comparative modeling of Balat_0669 and molecular docking studies support its role in chlorogenic acid hydrolysis. Expression, purification, and functional characterization of Balat_0669 in Escherichia coli were obtained as further validation. A possible role of B. animalis in the activation of hydroxycinnamic acids was demonstrated and new perspectives were opened in the development of new probiotics, specifically selected for the enhanced bioconversion of phytochemicals into bioactive compounds.
ESTHER : Raimondi_2015_Microbiolopen_4_41
PubMedSearch : Raimondi_2015_Microbiolopen_4_41
PubMedID: 25515139
Gene_locus related to this paper: bifa0-b8du06

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 : Muscarinic receptors as model targets and antitargets for structure-based ligand discovery - Kruse_2013_Mol.Pharmacol_84_528
Author(s) : Kruse AC , Weiss DR , Rossi M , Hu J , Hu K , Eitel K , Gmeiner P , Wess J , Kobilka BK , Shoichet BK
Ref : Molecular Pharmacology , 84 :528 , 2013
Abstract : G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse beta-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.
ESTHER : Kruse_2013_Mol.Pharmacol_84_528
PubMedSearch : Kruse_2013_Mol.Pharmacol_84_528
PubMedID: 23887926

Title : Identification and characterisation of a novel acylpeptide hydrolase from Sulfolobus solfataricus: structural and functional insights - Gogliettino_2012_PLoS.One_7_e37921
Author(s) : Gogliettino M , Balestrieri M , Cocca E , Mucerino S , Rossi M , Petrillo M , Mazzella E , Palmieri G
Ref : PLoS ONE , 7 :e37921 , 2012
Abstract : A novel acylpeptide hydrolase, named APEH-3(Ss), was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a "true" aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3(Ss) and apeh(Ss) (the gene encoding the previously identified APEH(Ss) from S. solfataricus), which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.
ESTHER : Gogliettino_2012_PLoS.One_7_e37921
PubMedSearch : Gogliettino_2012_PLoS.One_7_e37921
PubMedID: 22655081

Title : Regulation of M(3) muscarinic receptor expression and function by transmembrane protein 147 - Rosemond_2011_Mol.Pharmacol_79_251
Author(s) : Rosemond E , Rossi M , McMillin SM , Scarselli M , Donaldson JG , Wess J
Ref : Molecular Pharmacology , 79 :251 , 2011
Abstract : The M(3) muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions. To identify novel M3R-interacting proteins, we used a recently developed yeast two-hybrid screen (split ubiquitin method) to detect interactions among membrane proteins. This screen led to the identification of many novel M3R-associated proteins, including the putative membrane protein transmembrane protein 147 (Tmem147). The amino acid sequence of Tmem147 is highly conserved among mammals, but its physiological roles are unknown at present. We initially demonstrated that Tmem147 could be coimmunoprecipitated with M3Rs in cotransfected mammalian cells (COS-7 cells). Confocal imaging studies showed that Tmem147 was localized to endoplasmic reticulum (ER) membranes and that the Tmem147/M3R interaction occurred in the ER of cotransfected COS-7 cells, resulting in impaired trafficking of the M3R to the cell surface. To study the role of Tmem147 in modulating M3R function in a more physiologically relevant setting, we carried out studies with H508 human colon cancer cells that endogenously express M3Rs and Tmem147. Treatment of H508 cells with carbachol, a hydrolytically stable acetylcholine analog, promoted H508 cell proliferation and activation of the mitogenic kinase, p90RSK. Small interfering RNA-mediated knockdown of Tmem147 expression significantly augmented the stimulatory effects of carbachol on H508 cell proliferation and p90RSK activation. These effects were associated with an increase in the density of cell surface M3Rs. Our data clearly indicate that Tmem147 represents a potent negative regulator of M3R function, most likely by interacting with M3Rs in an intracellular compartment (ER). These findings may lead to new strategies aimed at modulating M3R activity for therapeutic purposes.
ESTHER : Rosemond_2011_Mol.Pharmacol_79_251
PubMedSearch : Rosemond_2011_Mol.Pharmacol_79_251
PubMedID: 21056967

Title : Genome sequence of Helicobacter bizzozeronii strain CIII-1, an isolate from human gastric mucosa - Schott_2011_J.Bacteriol_193_4565
Author(s) : Schott T , Rossi M , Hanninen ML
Ref : Journal of Bacteriology , 193 :4565 , 2011
Abstract : The canine-adapted Helicobacter bizzozeronii is the only nonpylori Helicobacter species isolated from human gastric biopsy tissue. Here we present the genome sequence of strain CIII-1, isolated from a 45-year-old female patient with severe gastric symptoms. This is the first genome sequence of nonpylori gastric Helicobacter isolated from human gastritis.
ESTHER : Schott_2011_J.Bacteriol_193_4565
PubMedSearch : Schott_2011_J.Bacteriol_193_4565
PubMedID: 21705603
Gene_locus related to this paper: helbc-f8krk7

Title : An atypical cutaneous reaction to rivastigmine transdermal patch - Grieco_2011_J.Allergy.(Cairo)_2011_752098
Author(s) : Grieco T , Rossi M , Faina V , De Marco I , Pigatto P , Calvieri S
Ref : J Allergy (Cairo) , 2011 :752098 , 2011
Abstract : Rivastigmine is a cholinesterase inhibitor which improves cognitive function and is currently being used in patients with mild to moderate Parkinson's and Alzheimer's dementia. This drug can be given orally or topically, as transdermal patch. The latter form is currently used for most excellent compliance and few side effects. The most common cutaneous side effects are irritative dermatitis. We report the second case of active sensitization by the rivastigmine-patch in a patient suffering from Alzheimer's dementia.
ESTHER : Grieco_2011_J.Allergy.(Cairo)_2011_752098
PubMedSearch : Grieco_2011_J.Allergy.(Cairo)_2011_752098
PubMedID: 21274348

Title : Genetic dissection of vitamin E biosynthesis in tomato - Almeida_2011_J.Exp.Bot_62_3781
Author(s) : Almeida J , Quadrana L , Asis R , Setta N , de Godoy F , Bermudez L , Otaiza SN , Correa da Silva JV , Fernie AR , Carrari F , Rossi M
Ref : J Exp Bot , 62 :3781 , 2011
Abstract : Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for alpha-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (alpha, beta, gamma, and delta) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.
ESTHER : Almeida_2011_J.Exp.Bot_62_3781
PubMedSearch : Almeida_2011_J.Exp.Bot_62_3781
PubMedID: 21527625
Gene_locus related to this paper: sollc-k4c685

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 : A novel class of protease targets of phosphatidylethanolamine-binding proteins (PEBP): a study of the acylpeptide hydrolase and the PEBP inhibitor from the archaeon Sulfolobus solfataricus - Palmieri_2010_Mol.Biosyst_6_2498
Author(s) : Palmieri G , Langella E , Gogliettino M , Saviano M , Pocsfalvi G , Rossi M
Ref : Mol Biosyst , 6 :2498 , 2010
Abstract : This work describes the identification and characterization of a Sulfolobus solfataricus acylpeptide hydrolase, named APEH(Ss), recognised as a new protease target of the endogenous PEBP inhibitor, SsCEI. APEH is one of the four members of the prolyl oligopeptidase (POP) family, which removes acylated amino acid residues from the N terminus of oligopeptides. APEH(Ss) is a cytosolic homodimeric protein with a molecular mass of 125 kDa. It displays a similar exopeptidase and endopeptidase activity to the homologous enzymes from Aeropyrum pernix and Pyrococcus horikoshii. Herein we demonstrate that SsCEI is the first PEBP protein found to efficiently inhibit APEH from both S. solfataricus and mammalian sources with IC(50) values in the nanomolar range. The 3D model of APEH(Ss) shows the typical structural features of the POP family including an N-terminal beta-propeller and a C-terminal alpha/beta hydrolase domain. Moreover, to gain insights into the binding mode of SsCEI toward APEH(Ss), a structural model of the inhibition complex is proposed, suggesting a mechanism of steric blockage on substrate access to the active site or on product release. Like other POP enzymes, APEH may constitute a new therapeutic target for the treatment of a number of pathologies and this study may represent a starting point for further medical research.
ESTHER : Palmieri_2010_Mol.Biosyst_6_2498
PubMedSearch : Palmieri_2010_Mol.Biosyst_6_2498
PubMedID: 20941418

Title : First Archaeal PEPB-Serine Protease Inhibitor from Sulfolobus solfataricus with Noncanonical Amino Acid Sequence in the Reactive-Site Loop - Palmieri_2009_J.Proteome.Res_8_327
Author(s) : Palmieri G , Catara G , Saviano M , Langella E , Gogliettino M , Rossi M
Ref : J Proteome Res , 8 :327 , 2009
Abstract : The specific inhibition of serine proteinases, which are crucial switches in many important physiological processes, is of great value both for basic research and for therapeutic applications. In this study, we report the molecular cloning of the sso0767 gene from Sulfolobus solfataricus, and the functional characterization of its product, SsCEI, which represents the first archaeal phosphatidylethanolamine-binding protein (PEBP)-serine proteinase inhibitor, reported to date. SsCEI is a monomer protein with a molecular mass of 19.0 kDa and a pI of 6.7, which is able to inhibit the serine proteases alpha-chymotrypsin and elastase with K(i) values of 0.08 and 0.1 microM, respectively. Moreover SsCEI is extremely resistant to both thermal inactivation and proteolytic attack suggesting compact folding of the protein. Within the I51 family, the archaeal inhibitor shows strong similarity to the human and murine members. The three-dimensional model of SsCEI revealed a general beta-fold and the presence of an anion-binding pocket, the hallmark of the PEBP family. Moreover SsCEI binds the cognate proteases according to a common, substrate-like standard mechanism. Point mutation experiments supported the prediction of the protease-binding site located on the surface at the C- terminal region of the protein. Interestingly, searches based on preidentified structural reactive loop motifs revealed the occurrence of a sequence (T123-N130) that is not represented in all serine-protease inhibitor families. This unique motif may provide new insights into both the inhibitor/protease binding mode and the specific biological functions of SsCEI within the PEBP family.
ESTHER : Palmieri_2009_J.Proteome.Res_8_327
PubMedSearch : Palmieri_2009_J.Proteome.Res_8_327
PubMedID: 19118453

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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : A novel thermophilic fusion enzyme for trehalose production - de Pascale_2002_Extremophiles_6_463
Author(s) : de Pascale D , Di Lernia I , Sasso MP , Furia A , De Rosa M , Rossi M
Ref : Extremophiles , 6 :463 , 2002
Abstract : In recent years a number of hyperthermophilic micro-organisms of Sulfolobales have been found to produce trehalose from starch and dextrins. In our laboratory genes encoding the trehalosyl dextrin forming enzyme (TDFE) and the trehalose forming enzyme (TFE) of S. solfataricus MT4 have been cloned and expressed in E. coli (Rb791). Here we report the construction of a new protein obtained by fusion of TFE and TDFE coding sequences which is able to produce trehalose from dextrins at high temperature by sequential enzymatic steps. We demonstrate that the bifunctional fusion enzyme is able to produce trehalose starting from malto-oligosaccharides at 75 degrees C. Furthermore we partially purified the recombinant fusion protein from bacterial cell free extracts and from insoluble fractions in which the fusion protein was also found as aggregate in inclusion bodies.
ESTHER : de Pascale_2002_Extremophiles_6_463
PubMedSearch : de Pascale_2002_Extremophiles_6_463
PubMedID: 12486454

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 : A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein - Morana_2002_Gene_283_107
Author(s) : Morana A , Di Prizito N , Aurilia V , Rossi M , Cannio R
Ref : Gene , 283 :107 , 2002
Abstract : A genomic library of the hyperthermophilic archaeon Sulfolobus solfataricus strain MT4 was constructed in Escherichia coli using a cloning vector not designed for heterologous gene expression. One positive clone exhibiting acquired thermophilic acetylesterase activity was directly detected by an in situ plate assay using a colony staining procedure with the chromogenic substrate beta-naphthyl acetate. The plasmid isolated from the clone contained a 3.3 kb genomic fragment from S. solfataricus and a full-length esterase coding sequence could be identified. Expression of the active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside and of the kind of vector, suggesting that the archaeal esterase gene was controlled by fortuitous bacterial-like sequences present in its own 5' flanking region, not by the bacterial lac promoter or other serendipitous vector-located sequences. The protein, partially purified by thermoprecipitation of the host proteins at high temperature and gel exclusion chromatography, showed a homo-tetrameric structure with a subunit of molecular mass of 32 kDa which was in perfect agreement with that deduced from the cloned gene. The same protein was revealed in S. solfataricus cell extracts, thus demonstrating its functional occurrence in vivo under the cell culture conditions tested. The recombinant enzyme exhibited high thermal activity and thermostability with optimal activity between pH 6.5 and 7.0. The hydrolysis of p-nitrophenyl esters of fatty acids (from C(2) to C(8)) allowed the enzyme to be classified as a short length acyl esterase.
ESTHER : Morana_2002_Gene_283_107
PubMedSearch : Morana_2002_Gene_283_107
PubMedID: 11867217
Gene_locus related to this paper: sulso-EstA

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 : 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 : 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 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 : 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 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