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Erwin Schneider - One of the best experts on this subject based on the ideXlab platform.
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structure of the atpase subunit cysa of the putative sulfate atp binding cassette abc transporter from Alicyclobacillus acidocaldarius
FEBS Letters, 2005Co-Authors: Frank Scheffel, Erwin Schneider, Ulrike Demmer, Eberhard Warkentin, Anja Hulsmann, Ulrich ErmlerAbstract:CysA, the ATPase subunit of a putative sulfate ATP-binding cassette transport system of the gram-positive thermoacidophilic bacterium Alicyclobacillus acidocaldarius, was structurally characterized at a resolution of 2.0 A in the absence of nucleotides. In line with previous findings on ABC-ATPases the structures of the two monomers (called CysA-1 and CysA-2) in the asymmetric unit differ substantially in the arrangement of their individual (sub)domains. CysA-2 was found as a physiological dimer composed of two crystallographically related monomers that are arranged in an open state. Interestingly, while the regulatory domain of CysA-2 packs against its opposing domain that of CysA-1 undergoes a conformational change and, in the dimer, would interfere with the opposing monomer thereby preventing solute translocation. Whether this conformational state is used for regulatory purposes will be discussed.
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Functional reconstitution of a maltose ATP-binding cassette transporter from the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius.
Biochimica et biophysica acta, 2004Co-Authors: Frank Scheffel, Rebecca Fleischer, Erwin SchneiderAbstract:Abstract The thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius grows at 60 °C and pH 2–3. The organism can utilize maltose and maltodextrins as energy source that are taken up by an ATP-binding cassette (ABC) import system. Genes encoding a maltose binding protein, MalE, and two membrane-integral subunits, MalF and MalG, are clustered on the chromosome but a malK gene translating into a cognate ATPase subunit is lacking. Here we report the cloning of malK from genomic DNA by using the msiK gene of Streptomyces lividans as a probe. Purified MalK exhibited a spontaneous ATPase activity with a Vmax of 0.13 μmol Pi/min/mg and a Km of 330 μM that was optimal at the growth temperature of the organism. Coexpression of malK, malF and malG in Escherichia coli resulted in the formation of a complex that could be coeluted from an affinity matrix after solubilization of membranes with dodecylmaltoside. Proteoliposomes prepared from the MalFGK complex and preformed phospholipid vesicles of A. acidocaldarius displayed a low intrinsic ATPase activity that was stimulated sevenfold by maltose-loaded MalE, thereby indicating coupling of ATP hydrolysis to substrate translocation. These results provide evidence for MalK being the physiological ATPase subunit of the A. acidocaldarius maltose transporter. Moreover, to our knowledge, this is the first report on the functional reconstitution of an ABC transport system from a thermophilic microorganism.
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x ray structures of the maltose maltodextrin binding protein of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius provide insight into acid stability of proteins
Journal of Molecular Biology, 2003Co-Authors: Karsten Schafer, Erwin Schneider, Frank Scheffel, Anja Hulsmann, Ulrika Magnusson, Andre Schiefner, Mats Sandgren, Kay Diederichs, Wolfram Welte, Sherry L MowbrayAbstract:Maltose-binding proteins act as primary receptors in bacterial transport and chemotaxis systems. We report here crystal structures of the thermoacidostable maltose-binding protein from Alicyclobacillus acidocaldarius, and explore its modes of binding to maltose and maltotriose. Further, comparison with the structures of related proteins from Escherichia coli (a mesophile), and two hyperthermophiles (Pyrococcus furiosus and Thermococcus litoralis) allows an investigation of the basis of thermo- and acidostability in this family of proteins. The thermoacidophilic protein has fewer charged residues than the other three structures, which is compensated by an increase in the number of polar residues. Although the content of acidic and basic residues is approximately equal, more basic residues are exposed on its surface whereas most acidic residues are buried in the interior. As a consequence, this protein has a highly positive surface charge. Fewer salt bridges are buried than in the other MBP structures, but the number exposed on its surface does not appear to be unusual. These features appear to be correlated with the acidostability of the A. acidocaldarius protein rather than its thermostability. An analysis of cavities within the proteins shows that the extremophile proteins are more closely packed than the mesophilic one. Proline content is slightly higher in the hyperthermophiles and thermoacidophiles than in mesophiles, and this amino acid is more common at the second position of b-turns, properties that are also probably related to thermostability. Secondary structural content does not vary greatly in the different structures, and so is not a contributing factor. q 2003 Elsevier Ltd. All rights reserved.
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a thermoacidophilic endoglucanase celb from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases
FEBS Journal, 2003Co-Authors: Kelvin Eckert, Erwin SchneiderAbstract:A 100-kDa protein with endoglucanase activity was purified from Triton X-100 extract of cells of the thermoacidophilic Gram-positive bacterium Alicyclobacillus acidocaldarius. The enzyme exhibited activity towards carboxy methyl cellulose and oat spelt xylan with pH and temperature optima of 4 and 80 °C, respectively. Cloning and nucleotide sequence analysis of the corresponding gene (celB) revealed an ORF encoding a preprotein of 959 amino acids which is consistent with an extracellular localization. Purified recombinant CelB and a variant lacking the C-terminal 203 amino acid residues (CelBtrunc) displayed similar enzymatic properties as the wild-type protein. Analysis of product formation suggested an endo mode of action. Remarkable stability was observed at pH values between 1 and 7 and 60% of activity were retained after incubation for 1 h at 80 °C. CelB displayed homology to members of glycoside hydrolase family 51, being only the second entry with activity typical of an endoglucanase but lacking activity on p-nitrophenyl-α-l-arabinofuranoside (pNPAraf). Highest sequence similarity was found towards the other endoglucanase F from Fibrobacter succinogenes (EGF), forming a distinct group in the phylogenetic tree of this family. Analysis of the amino acid composition of the catalytic domains demonstrated that CelB contains fewer charged amino acids than its neutrophilic counterparts, which is in line with adaptation to low pH. Wild-type and full-length recombinant CelB were soluble only in Triton X-100. In contrast, CelBtrunc was completely water soluble, suggesting a role of the C-terminal region in cell association. This C-terminal hydrophobic region displayed local sequence similarities to an α-amylase from the same organism.
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Crystallization and preliminary X-ray analysis of Alicyclobacillus acidocaldarius endoglucanase CelA.
Acta Crystallographica Section D Biological Crystallography, 2002Co-Authors: Kelvin Eckert, Heidi A Ernst, Erwin Schneider, Sine Larsen, Leila Lo LeggioAbstract:Crystallization of a family 9 β-1,4-glucanase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius CelA is reported. Thin plates can be obtained by hanging-drop vapour-diffusion crystallization in high concentrations (60%) of MPD. These crystals are unusual in that they do not bind the dye IZIT in the mother liquor and do not appear to dissolve in water after three weeks or in the storage buffer after 2 d. The crystals diffract weakly and the diffraction pattern is compatible with crystal disorder in one direction. After testing several crystals at the ESRF beamlines ID14-1 and ID14-2, a crystal was found which gave ordered diffraction in all directions. A full data set was collected to 3.0 A resolution, which allowed unambiguous determination of the space group as P21212 and the unit-cell parameters as a = 85, b = 129.7, c = 48.6 A. Initial promising results from molecular-replacement searches are reported.
Giuseppe Manco - One of the best experts on this subject based on the ideXlab platform.
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Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius
Extremophiles, 2015Co-Authors: Angela Pennacchio, Giuseppe Manco, Luigi Mandrich, Antonio TrinconeAbstract: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 α- 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 β- 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.
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Comprehensive analysis of surface charged residues involved in thermal stability in Alicyclobacillus acidocaldarius esterase 2.
Protein engineering design & selection : PEDS, 2012Co-Authors: Margherita Pezzullo, Luigi Mandrich, Pompea Del Vecchio, Mosè Rossi, Roberto Nucci, Giuseppe MancoAbstract: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 ΔΔG 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.
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Thermostable esterase 2 from Alicyclobacillus acidocaldarius as biosensor for the detection of organophosphate pesticides.
Analytical Chemistry, 2011Co-Authors: Ferdinando Febbraio, Luigia Merone, Giovanni Paolo Cetrangolo, Mosè Rossi, Roberto Nucci, Giuseppe MancoAbstract:Pesticides are the plague of modern times, although much needed in agriculture, causing damage to the entire ecosystem, including humans. The high operative costs and the requirement of specialized personnel for pesticide detection, incentive to develop alternative solutions such as the set up of cheap, rapid, and simple to use biosensors. In this work, we evaluate the possibility to use the esterase 2 from Alicyclobacillus acidocaldarius as a biosensor for the detection of specific organophosphate pesticides. With the recent demonstration of the very high affinity of esterase 2 toward paraoxon, a more complete analysis on the detection methods in water as well as in purposely contaminated fruit juices was carried out. The inhibitory effects of a wide range of other pesticides on esterase 2 were investigated, showing a better selectivity with respect to nonspecific reaction of acethylcholinesterases, the main target of organophosphate pesticides. The applied methodology allowed one to detect 2.75 × 10−3 p...
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Structural and kinetic overview of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius: a comparison with the other members of the HSL family.
Protein and peptide letters, 2009Co-Authors: Giuseppe Manco, Luigia Merone, Luigi MandrichAbstract: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.
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Irreversible inhibition of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius
Extremophiles, 2008Co-Authors: Ferdinando Febbraio, Luigia Merone, Luigi Mandrich, Mosè Rossi, Roberto Nucci, Sandro Esposito D’andrea, Giuseppe MancoAbstract: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.
Mosè Rossi - One of the best experts on this subject based on the ideXlab platform.
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Comprehensive analysis of surface charged residues involved in thermal stability in Alicyclobacillus acidocaldarius esterase 2.
Protein engineering design & selection : PEDS, 2012Co-Authors: Margherita Pezzullo, Luigi Mandrich, Pompea Del Vecchio, Mosè Rossi, Roberto Nucci, Giuseppe MancoAbstract: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 ΔΔG 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.
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Thermostable esterase 2 from Alicyclobacillus acidocaldarius as biosensor for the detection of organophosphate pesticides.
Analytical Chemistry, 2011Co-Authors: Ferdinando Febbraio, Luigia Merone, Giovanni Paolo Cetrangolo, Mosè Rossi, Roberto Nucci, Giuseppe MancoAbstract:Pesticides are the plague of modern times, although much needed in agriculture, causing damage to the entire ecosystem, including humans. The high operative costs and the requirement of specialized personnel for pesticide detection, incentive to develop alternative solutions such as the set up of cheap, rapid, and simple to use biosensors. In this work, we evaluate the possibility to use the esterase 2 from Alicyclobacillus acidocaldarius as a biosensor for the detection of specific organophosphate pesticides. With the recent demonstration of the very high affinity of esterase 2 toward paraoxon, a more complete analysis on the detection methods in water as well as in purposely contaminated fruit juices was carried out. The inhibitory effects of a wide range of other pesticides on esterase 2 were investigated, showing a better selectivity with respect to nonspecific reaction of acethylcholinesterases, the main target of organophosphate pesticides. The applied methodology allowed one to detect 2.75 × 10−3 p...
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Irreversible inhibition of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius
Extremophiles, 2008Co-Authors: Ferdinando Febbraio, Luigia Merone, Luigi Mandrich, Mosè Rossi, Roberto Nucci, Sandro Esposito D’andrea, Giuseppe MancoAbstract: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.
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isolation and characterization of a new family 42 β galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius identification of the active site residues
Biochimica et Biophysica Acta, 2008Co-Authors: Barbara Di Lauro, Mosè Rossi, Andrea Strazzulli, Giuseppe Perugino, Francesco La Cara, Emiliano Bedini, Maria Michela Corsaro, Marco MoracciAbstract:Abstract The thermoacidophilic bacterium Alicyclobacillus acidocaldarius is a rich source of glycoside hydrolases enabling its growth on several di- and polysaccharides. We report here the purification and the characterization of a β-galactosidase from this source, the cloning of its gene, and the expression and the characterization of the recombinant enzyme (Aaβ-gal).The enzyme was purified 46-fold from A. acidocaldarius extracts; the gene for Aaβ-gal encoded a new member of the glycoside hydrolase family 42 (GH42) and it is flanked by a putative AraC/XylS regulator, however, the two genes were transcribed independently. The recombinant Aaβ-gal was characterized in detail revealing that it is optimally active and stable at 65 °C. Aaβ-gal is very specific for glycosides with an axial C4-OH at their non-reducing end, with k cat / K M values of 484, 186, and 332 s − 1 mM − 1 for 2-nitrophenyl-β- d -galactoside, -fucoside, and 4-nitrophenyl-α- l -arabinoside, respectively. Finally, the characterization of the site-directed mutants Glu157Gly and Glu313Gly confirmed the latter as the nucleophile of the reaction and gave experimental evidence, for the first time in GH42, of the role of Glu157 as the acid/base of the catalyzed reaction.
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functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius
Proteins, 2007Co-Authors: Luigi Mandrich, Giuseppina De Simone, Valeria Menchise, Carlo Pedone, Mosè Rossi, Vincenzo Alterio, Giuseppe MancoAbstract: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°C and pH = 5.75). The catalytic constants K M and Vmax were found considerably altered in the mutant, with ninefold increased K M and 14-fold decreased Vmax, 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. Proteins 2008. © 2007 Wiley-Liss, Inc.
Karl Poralla - One of the best experts on this subject based on the ideXlab platform.
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Specific production of γ-polypodatetraene or 17-isodammara-20(21),24-diene by squalene–hopene cyclase mutant
Tetrahedron Letters, 2001Co-Authors: Susanne Schmitz, Christine Füll, Tobias Glaser, Klaus Albert, Karl PorallaAbstract:Amino acids lining the catalytic cavity of squalene–hopene cyclase of Alicyclobacillus acidocaldarius were mutated to investigate their catalytic functions. Mutagenesis of Leu607 to Lys in the central part of the cavity resulted in the production of the bicyclic γ-polypodatetraene (1) as main product, while the mutation of Phe605 to Lys near the deprotonation site of the cavity led mainly to the formation of tetracyclic 17-isodammara-20(21),24-diene (2).
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Conserved tyr residues determine functions of Alicyclobacillus acidocaldarius squalene-hopene cyclase.
FEMS microbiology letters, 2000Co-Authors: Christine Füll, Karl PorallaAbstract:The catalytic cavity of Alicyclobacillus acidocaldarius squalene–hopene cyclase is mainly lined by aromatic amino acids. In recombinant cyclases, three out of four tyrosine residues (Y) have been mutated to phenylalanine residues (F). The mutant cyclases Y495F and Y612F had less activity than the wild-type cyclase, but a wild-type product pattern. Mutant Y609F had wild-type activity but a drastically altered product pattern with hopene and significant amounts of bicyclic α-polypodatetraene and different tetracyclic triterpenes (dammaradienes and eupha-7,24-diene). The experiments demonstrated that Y495 and Y612 may be involved in the initiation of the cyclization reaction and Y609 in the stabilization and/or positioning of the intermediate carbocations.
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Production of bicyclic and tricyclic triterpenes by mutated squalene-hopene cyclase
Tetrahedron Letters, 1999Co-Authors: Catherine Pale-grosdemange, Thorsten Merkofer, Michel Rohmer, Karl PorallaAbstract:Mutagenesis of Tyr420 to Ala in the catalytic cavity of squalene-hopene cyclase of Alicyclobacillus acidocaldarius resulted in an altered product spectrum. Besides hopene and diplopterol, this mutant produced significant amounts of the bicyclic α- and γ-polypodatetraenes and minor amounts of the tricyclic 13α(H)-malabaricatriene.
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ALTERED PRODUCT PATTERN OF A SQUALENE-HOPENE CYCLASE BY MUTAGENESIS OF ACTIVE SITE RESIDUES
Tetrahedron Letters, 1999Co-Authors: Thorsten Merkofer, Catherine Pale-grosdemange, Michel Rohmer, Karl Ulrich Wendt, Karl PorallaAbstract:Amino acid residues lining the catalytic cavity of squalene-hopene cyclase of Alicyclobacillus acidocaldarius have been mutated. Alterations of His451 to Ala and Trp489 to Ala resulted in reduced enzymatic activity, while the product patterns were identical to that of the wild-type. Mutation of Phe601 to Ala led to the enhanced formation of a tetracyclic triterpene, 17-isodammara-20(21),24-diene 4, and of Tyr420 to Ala to a significant alteration of the product pattern.
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Zymomonas mobilis squalene-hopene cyclase gene (shc): cloning, DNA sequence analysis, and expression in Escherichia coli.
Microbiology, 1995Co-Authors: Ina G. Reipen, Hermann Sahm, Karl Poralla, Georg A SprengerAbstract:SUMMARY: Using a DNA probe from the gene encoding squalene-hopene cyclase (SHC, EC 5.4.99.-) from the Gram-positive bacterium Alicyclobacillus acidocaldarius, we have cloned a 4·3 kb Hindll fragment of chromosomal DNA from Zymomonas mobilis. An open reading frame of 1977 bp was detected that could encode a protein of 658 amino acids with a calculated molecular mass of 74077 Da. Under the control of lac or tac promoters, this gene, shc, was expressed in Escherichia coli K12 strains and its product had squalene-hopene cyclase activity. Sequence alignments with the A. acidocaldarius SHC, the lanosterol cyclase of the yeast Candida albicans, and the cycloartenol synthase of the plant Arabidopsis thaliana revealed six highly conserved regions (mainly in the C-terminal part) of the proteins. These regions contained the core motif Gln-X-X-X-Gly-X-Trp.
Mathias Sprinzl - One of the best experts on this subject based on the ideXlab platform.
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Detection of bacterial 16S rRNA using multivalent dendrimer-reporter enzyme conjugates
Biosensors & bioelectronics, 2009Co-Authors: Christopher Pöhlmann, Martin Humenik, Mathias SprinzlAbstract:Novel enzyme–oligodeoxynucleotide conjugate was synthesized to improve sensitivity of Escherichia coli 16S rRNA detection on gold electrodes. Thermostable esterase 2 from Alicyclobacillus acidocaldarius was multiply conjugated to a polyamidoamine dendrimer functionalized by one universal detector oligodeoxynucleotide. Three components rRNA/DNA hybridization between capture oligodeoxynucleotide covalently immobilized on a gold electrode, 16S rRNA and the multivalent esterase–dendrimer cluster was used for detection of E. coli. The linear dependence of the electrochemical signals to analyte concentration revealed a detection limit of 50 colony forming units E. coli, which represents a tenfold signal enhancement if compared to the detection limit achieved with monovalent esterase–oligodeoxynucleotide conjugate.
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Rapid, specific and sensitive electrochemical detection of foodborne bacteria
Biosensors & bioelectronics, 2009Co-Authors: Christopher Pöhlmann, Yiran Wang, Martin Humenik, Bernd Heidenreich, Manfred Gareis, Mathias SprinzlAbstract:Abstract Electrochemical biochips are an emerging tool for point-of-care diagnostic systems in medicine, food and environmental monitoring. In the current study, a thermostable reporter enzyme, esterase 2 (EST2) from Alicyclobacillus acidocaldarius , is used for specific and sensitive detection of bacteria by one-step rRNA/DNA hybridization between a bacterium-specific capture oligodeoxynucleotide (ODN), bacterial 16S rRNA and an uniform EST2–ODN reporter conjugate. The detection limit corresponds to approximately 500 colony forming units (cfu) Escherichia coli . Beside high sensitivity, the application of electrochemical biochips allows discrimination of two Gram-negative and two Gram-positive bacteria demonstrating the specificity and the potential for parallel detection of microorganisms. The feasibility of identification of foodborne bacteria was studied with meat juice contaminated with E. coli . This detection system has the capability to be applied for monitoring of bacterial food contamination.
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Enhancement of electrochemical signal on gold electrodes by polyvalent esterase-dendrimer clusters.
Bioconjugate chemistry, 2008Co-Authors: Martin Humenik, Christopher Pöhlmann, Yiran Wang, Mathias SprinzlAbstract:5'-Maleimide-oligodeoxynucleotide was conjugated with single sulfhydryl group of cystamine core poly(amidoamine) dendrimers of different generations. Amino groups on the dendrimer moiety were modified with maleimide and coupled to the cysteine 118 of esterase 2 from Alicyclobacillus acidocaldarius in a site-specific manner. Polyvalent esterase-dendrimer-oligodeoxynucleotide clusters were hybridized to capture oligodeoxynucleotides immobilized on a gold electrode. The amperometric signal of p-aminophenol was detected following the esterase-catalyzed hydrolysis of p-aminophenylbutyrate. The multiple anchoring of the esterase reporter via generation 3-and generation 5-derived clusters exhibited 10- and 100-fold signal enhancement, respectively, as compared to monovalent esterase-oligonucleotide conjugate. The polyvalent and monovalent reporters were comparable in their abilities regarding mismatch discrimination.
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Ligand-directed immobilization of proteins through an esterase 2 fusion tag studied by atomic force microscopy.
Chembiochem : a European journal of chemical biology, 2008Co-Authors: Antonín Minařík, Martin Humenik, Yiwei Huang, Georg Krausch, Mathias SprinzlAbstract:Atomically flat mica surfaces were chemically modified with an alkyl trifluoromethyl ketone, a covalent inhibitor of esterase 2 from Alicyclobacillus acidocaldarius, which served as a tag for ligand-directed immobilization of esterase-linked proteins. Purified NADH oxidase from Thermus thermophilus and human exportin-t from cell lysates were anchored on the modified surfaces. The immobilization effectiveness of the proteins was studied by atomic force microscopy (AFM). It was shown that ligand-esterase interaction allowed specific attachment of exportin-t and resulted in high-resolution images and coverage patterns that were comparable with immobilized purified protein. Moreover, the biological functionality of immobilized human exportin-t in forming a quaternary complex with tRNA and the GTPase Ran-GTP, and the dimension changes before and after complex formation were also determined by AFM.
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Esterase 2 from Alicyclobacillus acidocaldarius as a reporter and affinity tag for expression and single step purification of polypeptides.
Protein expression and purification, 2007Co-Authors: Yiwei Huang, Martin Humenik, Mathias SprinzlAbstract:A novel dual function (reporter and affinity) tag system has been developed. Expression vectors have been constructed to express polypeptides in Escherichia coli cells as C-terminal fusions with esterase 2, a 34-kDa protein from Alicyclobacillus acidocaldarius. Presence of esterase allows to monitor the expression of fusion proteins spectrophotometrically or by activity staining in the polyacrylamide gels. The fusion proteins can be purified from crude bacterial extracts under non-denaturing conditions by one step affinity chromatography on Sepharose CL-6B immobilized trifluoromethyl-alkyl-ketone. The esterase carrier can be cleaved from fusion proteins by digestion with amino acid sequence-specific proteases blood coagulation factor Xa. The system has been used successfully for the expression and purification of polypeptides from different prokaryotic and eukaryotic organisms.
