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Native Enzyme (native + enzyme)
Selected AbstractsRedox properties of the couple compound I/native enzyme of myeloperoxidase and eosinophil peroxidaseFEBS JOURNAL, Issue 19 2001Jürgen Arnhold The standard reduction potential of the redox couple compound I/native enzyme has been determined for human myeloperoxidase (MPO) and eosinophil peroxidase (EPO) at pH 7.0 and 25 °C. This was achieved by rapid mixing of peroxidases with either hydrogen peroxide or hypochlorous acid and measuring spectrophotometrically concentrations of the reacting species and products at equilibrium. By using hydrogen peroxide, the standard reduction potential at pH 7.0 and 25 °C was 1.16 ± 0.01 V for MPO and 1.10 ± 0.01 V for EPO, independently of the concentration of hydrogen peroxide and peroxidases. In the case of hypochlorous acid, standard reduction potentials were dependent on the hypochlorous acid concentration used. They ranged from 1.16 V at low hypochlorous acid to 1.09 V at higher hypochlorous acid for MPO and from 1.10 V to 1.03 V for EPO. Thus, consistent results for the standard reduction potentials of redox couple compound I/native enzyme of both peroxidases were obtained with all hydrogen peroxide and at low hypochlorous acid concentrations: possible reasons for the deviation at higher concentrations of hypochlorous acid are discussed. They include instability of hypochlorous acid, reactions of hypochlorous acid with different amino-acid side chains in peroxidases as well as the appearance of a compound I,chloride complex. [source] Chemical and enzymatic hydrolysis of anthraquinone glycosides from madder rootsPHYTOCHEMICAL ANALYSIS, Issue 3 2003Goverdina C. H. Derksen Abstract For the production of a commercially useful dye extract from madder, the glycoside ruberythric acid has to be hydrolysed to the aglycone alizarin which is the main dye component. An intrinsic problem is the simultaneous hydrolysis of the glycoside lucidin primeveroside to the unwanted mutagenic aglycone lucidin. Madder root was treated with strong acid, strong base or enzymes to convert ruberythric acid into alizarin and the anthraquinone compositions of the suspensions were analysed by HPLC. A cheap and easy method to hydrolyse ruberythric acid in madder root to alizarin without the formation of lucidin turned out to be the stirring of dried madder roots in water at room temperature for 90 min: this gave a suspension containing pseudopurpurin, munjistin, alizarin and nordamnacanthal. Native enzymes are responsible for the hydrolysis, after which lucidin is converted to nordamnacanthal by an endogenous oxidase. Copyright © 2003 John Wiley & Sons, Ltd. [source] The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitiveFEBS JOURNAL, Issue 20 2004Linda O'Reilly Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the ,-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady-state levels of the Y42H variant in comparison to wild-type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures. [source] Engineering of a monomeric and low-glycosylated form of human butyrylcholinesteraseFEBS JOURNAL, Issue 2 2002Expression, characterization, crystallization, purification Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 Å resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 Å, c = 124.9 Å, giving a Vm of 2.73 Å3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general. [source] Enzyme-mediated sulfide production for the reconstitution of [2Fe,2S] clusters into apo-biotin synthase of Escherichia coliFEBS JOURNAL, Issue 9 2000Sulfide transfer from cysteine to biotin We previously showed that biotin synthase in which the (Fe,S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Méjean & A. Marquet (1998) FEBS Lett. 440, 226,230]. We therefore proposed that the source of sulfur was very likely the (Fe,S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated. In this report, we show that the NifS protein from Azotobacter vinelandii and C-DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe,2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme. When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S-reconstituted enzyme. NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe,Se) cluster. However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay. [source] Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxideFEBS JOURNAL, Issue 5 2000Evidence for a dithiol, disulfide equilibrium, implications for redox regulation Calcineurin (CaN) is a Ca2+ -and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe,Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3,8 µm and the inactivation was reversed by 2,3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+,Zn2+ dinuclear centre. Upon H2O2 -mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN. [source] Purification and characterization of a glutathione S -transferase from the fungus Cunninghamella elegansFEMS MICROBIOLOGY LETTERS, Issue 2 2001Chang-Jun Cha Abstract Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S -transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120,000×g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate,polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of Mr 27,000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian ,-, ,-, and ,-class GSTs, although it showed a small degree of cross-reactivity with a ,-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs. [source] Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilizationJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 5 2001Jesús Torres-Bacete Abstract Penicillin,V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy-activated acrylic beads (Eupergit C®) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55,°C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10-fold increase in its half-life at 40,°C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation. © 2001 Society of Chemical Industry [source] Cerebrotendinous xanthomatosis: molecular characterization of two Scandinavian sistersJOURNAL OF INTERNAL MEDICINE, Issue 3 2002E. Rystedt Abstract.,Rystedt E, Olin M, Seyama Y, Buchmann M, Berstad A, Eggertsen G, Björkhem I (Karolinska Institutet, Stockholm, Sweden; OchanomizuUniversity, Tokyo, Japan; Medisinsk avdeling, Haukeland sykehus, Bergen). Cerebrotendinous xanthomatosis: molecular characterization of two Scandinavian sisters (Case report). Journal of Internal Medicine 2002; 252: 259,264. Cerebrotendinous xanthomatosis (CTX) is a hereditary disorder, which is inherited as an autosomally recessive disease, causing production of cholesterol and cholestanol xanthomas and mental retardation. The disease is caused by mutations in the gene for sterol 27-hydroxylase (CYP27A1). The only CTX patients diagnosed in Scandinavia are two Norwegian sisters from a consanguineous marriage. Here we have characterized the mutation and its functional consequences for the enzyme. Analysis of genomic DNA from cultured fibroblasts identified a base exchange C > T in position 1441, causing arginine at amino acid position 441 to be replaced by tryptophan. The same mutation was introduced by mutagenesis in the complimentary DNA (cDNA) for CYP27, ligated into the expression vector pcDNA4/HisMax and transfected into HEK293 cells. The mutated enzyme had less than 5% of the enzyme activity compared with the native enzyme. No abnormal catalytic products could be identified in the cell culture medium. Probably the mutation affects the haem binding within the holoenzyme. The mutation has also previously been reported in a Japanese family. This is the second example of a CTX-causing mutation that has been recognized in more than one population. [source] Detection of the Sm31 antigen in sera of Schistosoma mansoni, infected patients from a low endemic areaPARASITE IMMUNOLOGY, Issue 1 2010G. S. SULBARÁN Summary Schistosoma mansoni cathepsin B (Sm31) is a major antigen from adult worms that circulates in the blood of infected patients (Li et al., Parasitol Res 1996; 82: 14,18). An analysis of the Sm31 sequence (Klinkert et al., Mol Biochem Parasitol 1989; 33: 113,122) allowed the prediction of seven hydrophilic regions that were confirmed to be exposed on the surface of a 3D model of Sm31; the species specificity of these regions was checked using BLAST analysis. The corresponding peptides were chemically synthesized in polymerazible forms using the t-Boc technique. Rabbits developed a high humoral response against these peptides as tested by a multiple antigen blot assay; it recognized native Sm31 in crude S. mansoni extracts and as circulating antigen in sera of S. mansoni-infected patients by western blot. Relevant antigenic determinants were located at the N- and C-terminus sequences. Antibodies against these regions recognized the native enzyme in an ELISA-like assay called cysteine protease immuno assay in which the immunocaptured enzyme was revealed by the intrinsic cathepsin B hydrolytic activity of Sm31. The method successfully and specifically detected Sm31 in sera of infected individuals, most of them (83·3%) with light infections, offering a rationale for the development of parasite enzyme capture assays using anti-synthetic peptide antibodies for possible use in the diagnosis of schistoso,iasis. [source] Bifunctional indole-3-acetyl transferase catalyses synthesis and hydrolysis of indole-3-acetyl- myo -inositol in immature endosperm of Zea maysPHYSIOLOGIA PLANTARUM, Issue 2 2003Stanislaw Kowalczyk 1- O -(indole-3-acetyl)- , - d -glucose: myo -inositol indoleacetyl transferase (IA- myo -inositol synthase) is an important enzyme in IAA metabolism. This enzyme catalyses the transfer of the indole acetyl (IA) moiety from 1- O -(indole-3-acetyl)- , - d -glucose to myo -inositol to form IA- myo- inositol and glucose. IA- myo -inositol synthase was purified to an electrophoretically homogenous state from maize liquid endosperm by fractionation with ammonium sulphate, anion-exchange, adsorption on hydroxylapatite, affinity chromatography on ConA-Sepharose, preparative PAGE and isoelectric focusing. We thus obtained two enzyme preparations which differ in their Rf on 8% polyacrylamide gel. The preparation of Rf 0.36 contained a single 56.4 kDa polypeptide, whereas the preparation of Rf 0.39 consisted of two polypeptides of 56.4 and 53.5 kDa. Both purified preparations of IAInos synthase also exhibited the activity of an IAInos hydrolase, showing that the dual activity was associated with a single protein. Results of gel filtration and analytical SDS-PAGE suggest that the native enzyme exists as both a monomeric (65 kDa) and homo- or heterodimeric form (110,130 kDa). Analysis of peptide maps and amino acid sequences of two 21 amino-acid peptides showed that polypeptides of 56.4 and 53.5 kDa have the same primary structure and that the 3 kDa difference in molecular mass is probably caused by different glycosylation levels. Comparison of this partial and internal amino acid sequence with sequences of other plant acyltransferases indicated similarity to several proteins which belonged to the serine carboxypeptidase-like (SCPL) acyltransferase family. [source] Production and characterization of a recombinant beta-1,4-endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipesARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2010Xuguo Zhou Abstract Cell-1 is a host-derived beta-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell-1 using eukaryotic (Baculovirus Expression Vector System; BEVS) and prokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily obtained in solubilized form and more active than the E. coli,expressed enzyme. Km and Vmax values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993% w/v and 1.056,µmol/min/mg. Additional characterization studies on the BEVS-expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5,7.5 and 50,60°C, is inhibited by EDTA, and displays enhanced activity up to 70°C in the presence of CaCl2. These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol-oxidizing laccases, and others. © 2010 Wiley Periodicals, Inc. [source] Critical catalytic functional groups in the mechanism of aspartate-,-semialdehyde dehydrogenaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2004Julio Blanco Aspartate-,-semialdehyde dehydrogenase (ASADH) catalyzes the reductive dephosphorylation of ,-aspartyl phosphate to l -aspartate-,-semialdehyde in the aspartate biosynthetic pathway. This pathway is not found in humans or other eukaryotic organisms, yet is required for the production of threonine, isoleucine, methionine and lysine in most microorganisms. The mechanism of this enzyme has been examined through the structures of two active-site mutants of ASADH from Haemophilus influenzae. Replacement of the enzyme active-site cysteine with serine (C136S) leads to a dramatic loss of catalytic activity caused by the expected decrease in nucleophilicity, but also by a change in the orientation of the serine hydroxyl group relative to the cysteine thiolate. In contrast, in the H277N active-site mutant the introduced amide is oriented in virtually the same position as that of the histidine imidazole ring. However, a shift in the position of the bound reaction intermediate to accommodate this shorter asparagine side chain, coupled with the inability of this introduced amide to serve as a proton acceptor, results in a 100-fold decrease in the catalytic efficiency of H277N relative to the native enzyme. These mutant enzymes have the same overall fold and high structural identity to native ASADH. However, small perturbations in the positioning of essential catalytic groups or reactive intermediates have dramatic effects on catalytic efficiency. [source] A new crystal form of XT6 enables a significant improvement of its diffraction quality and resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Maya Bar Xylanases (1,4-,- d -xylan xylanhydrolases; EC 3.2.1.8) hydrolyze the 1,4-,- d -xylopyranosyl linkage of xylans. The detailed structural characterization of these enzymes is of interest for the elucidation of their catalytic mechanism and for their rational modification toward improved stability and specificity. An extracellular xylanase from Geobacillus stearothermophilus T-6 (XT6) has recently been cloned, overexpressed, purified and biochemically characterized. Previous crystallographic efforts resulted in a hexagonal crystal form, which subsequently proved to be of limited use for structural analysis, mainly because of its relatively poor diffraction quality and resolution. A systematic search for more suitable crystals of XT6 recently resulted in a new crystal form of this enzyme with significantly improved diffraction characteristics. The new crystals belong to a C -centred monoclinic crystal system (space group C2), with unit-cell parameters a = 121.5, b = 61.7, c = 89.1,Å, , = 119.7°. These crystals diffract X-rays to better than 1.5,Å resolution, showing a very clear diffraction pattern of relatively high quality. The crystals are mechanically strong and exhibit excellent radiation-stability when frozen under cold nitrogen gas. A full diffraction data set to 1.45,Å resolution (94.1% completeness, Rmerge = 7.0%) has been collected from flash-frozen crystals of the native enzyme at 95,K using synchrotron radiation. Crystals of the E159A/E265A catalytic double mutant of XT6 were found to be isomorphous to those of native XT6. They were used for a full measurement of 1.8,Å resolution diffraction data at 100,K (90.9% completeness; Rmerge = 5.0%). These data are currently being used for the high-resolution structure determination of XT6 and its mutant for mechanistic interpretations and rational introduction of thermostability. [source] Crystallization and preliminary crystallographic analysis of the recombinant N-terminal domain of riboflavin synthaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001Winfried Meining Riboflavin synthase catalyzes the final step in the biosynthesis of riboflavin. Animals and humans lack this enzyme, whereas many bacteria and certain yeasts are absolutely dependent on endogenous riboflavin synthesis. Riboflavin synthase is therefore an attractive target for chemotherapy. The N-terminal domain of riboflavin synthase forms a dimer in solution and is capable of strongly binding riboflavin. It can serve as a model for the binding site of the native enzyme. Structural information obtained from this domain at high resolution will be helpful in the determination of the binding mode of riboflavin and thus for the development of antimicrobial drugs. Here, the crystallization and preliminary crystallographic analysis of the N-terminal domain of riboflavin synthase are reported. The crystals belong to the space group C2221, with unit-cell parameters a = 50.3, b = 104.7, c = 85.3,Å, , = , = , = 90°, and diffract to 2.6,Å resolution. [source] Atomic resolution structure of Escherichia coli dUTPase determined ab initioACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2001A. González Cryocooled crystals of a mercury complex of Escherichia coli dUTPase diffract to atomic resolution. Data to 1.05,Å resolution were collected from a derivative crystal and the structure model was derived from a Fourier map with phases calculated from the coordinates of the Hg atom (one site per subunit of the trimeric enzyme) using the program ARP/wARP. After refinement with anisotropic temperature factors a highly accurate model of the bacterial dUTPase was obtained. Data to 1.45,Å from a native crystal were also collected and the 100,K structures were compared. Inspection of the refined models reveals that a large part of the dUTPase remains rather mobile upon freezing, with 14% of the main chain being totally disordered and with numerous side chains containing disordered atoms in multiple discrete conformations. A large number of those residues surround the active-site cavity. Two glycerol molecules (the cryosolvent) occupy the deoxyribose-binding site. Comparison between the native enzyme and the mercury complex shows that the active site is not adversely affected by the binding of mercury. An unexpected effect seems to be a stabilization of the crystal lattice by means of long-range interactions, making derivatization a potentially useful tool for further studies of inhibitor,substrate-analogue complexes of this protein at very high resolution. [source] Crystallization and initial crystallographic analysis of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosaACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2000Catherine A. Regni The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) catalyzes the conversion of mannose 6-phosphate to mannose 1-phosphate in the second step of the alginate biosynthetic pathway of Pseudomonas aeruginosa. PMM/PGM has been crystallized by hanging-drop vapor diffusion in space group P212121. Crystals diffract to 1.75,Å resolution on a synchrotron X-ray source under cryo-cooling conditions. PMM/PGM substituted with selenomethionine has been purified and crystallizes isomorphously with the native enzyme. Structure determination by MAD phasing is under way. Because of its role in alginate biosynthesis, PMM/PGM is a potential target for therapeutic inhibitors to combat P. aeruginosa infections. [source] Crystallization and preliminary crystallographic analysis of Thermus thermophilus leucyl-tRNA synthetase and its complexes with leucine and a non-hydrolysable leucyl-adenylate analogueACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2000Anna Yaremchuk Leucyl-tRNA synthetase from Thermus thermophilus (LeuRSTT) is the first LeuRS to be crystallized. Two crystal forms of the native enzyme have been obtained using the hanging-drop vapour-diffusion method with ammonium sulfate as a precipitant. Crystals of the first form belong to space group I422 and have unit-cell parameters a = b = 312.4, c = 100.4,Å. They diffract anisotropically to 3.5,Å resolution in the c -axis direction and to only 6,Å resolution in the perpendicular direction. Crystals of the second form, which can be obtained native or with leucine or a leucyl-adenylate analogue bound, belong to space group C2221 and have unit-cell parameters a = 102.4, b = 154.1, c = 174.3,Å. They diffract to 1.9,Å resolution and contain one monomer in the asymmetric unit. Selenomethionated LeuRSTT has been produced and crystals of the second form suitable for MAD analysis have been grown. [source] Isolation and purification of Thermus thermophilus HpaB by a crystallization approachACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Tewfik Soulimane The oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme. While isolating integral membrane respiratory complexes from Thermus thermophilus, microcrystals of HpaB were formed. Further purification of the enzyme was achieved by repetitive crystallization. Subsequently, well shaped single crystals of the native enzyme that diffract to 1.82,Å resolution were grown in sitting drops. They belong to the orthorhombic space group I222, with unit-cell parameters a = 91.3, b = 99.8, c = 131.7,Å. [source] Crystallization and preliminary X-ray diffraction analysis of inositol 1,3,4,5,6-pentakisphosphate kinase from Arabidopsis thalianaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Jose Ignacio Baños-Sanz Inositol 1,3,4,5,6-pentakisphosphate kinase (IP5 2-K) is an enzyme involved in inositol metabolism that synthesizes IP6 (inositol 1,2,3,4,5,6-hexakisphosphate) from inositol 1,3,4,5,6-pentakisphosphate (IP5) and ATP. IP6 is the major phosphorus reserve in plants, while in mammals it is involved in multiple cellular events such as DNA editing and chromatin remodelling. In addition, IP6 is the precursor of other highly phosphorylated inositols which also play highly relevant roles. IP5 2-K is the only enzyme that phosphorylates the 2-OH axial position of the inositide and understanding its molecular mechanism of substrate specificity is of great interest in cell biology. IP5 2-K from Arabidopsis thaliana has been expressed in Escherichia coli as two different fusion proteins and purified. Both protein preparations yielded crystals of different quality, always in the presence of IP6. The best crystals obtained for X-ray crystallographic analysis belonged to space group P212121, with unit-cell parameters a = 58.124, b = 113.591, c = 142.478,Å. Several diffraction data sets were collected for the native enzyme and two heavy-atom derivatives using a synchrotron source. [source] Expression, purification and crystallization of the cofactor-independent monooxygenase SnoaB from the nogalamycin biosynthetic pathwayACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Hanna Koskiniemi 12-deoxy-nogalonic acid oxygenase (SnoaB) catalyzes the oxygenation of 12-deoxy-nogalonic acid at position 12 to yield nogalonic acid, which is one of the steps in the biosynthesis of the polyketide nogalamycin in Streptomyces nogalater. SnoaB belongs to a family of small cofactor-free oxygenases which carry out oxygenation reactions without the aid of any prosthetic group, cofactor or metal ion. Recombinant SnoaB was crystallized in space group P21212, with unit-cell parameters a = 58.8, b = 114.1, c = 49.5,Å, and these crystals diffracted to 2.4,Å resolution. Recombinant SnoaB does not contain any methionine residues and three double mutants were designed and produced for the preparation of selenomethionine-substituted samples. The selenomethionine-substituted mutant F40M/L89M crystallized in the same space group as the native enzyme. [source] Stabilization of invertase by molecular engineeringBIOTECHNOLOGY PROGRESS, Issue 1 2010Pattamawadee Tananchai Abstract Extracellular invertase (EC 3.2.1.26) of Saccharomyces cerevisiae was stabilized against thermal denaturation by intermolecular and intramolecular crosslinking of the surface nucleophilic functional groups with diisocyanate homobifunctional reagents (OCN(CH2)nNCO) of various lengths (n = 4, 6, 8). Crosslinking with 1,4-diisocyanatobutane (n = 4) proved most effective in enhancing thermostability. Stability was improved dramatically by crosslinking 0.5 mg/mL of protein with 30 ,mol/mL of the reagent. Molecular engineering by crosslinking reduced the first-order thermal denaturation constant at 60°C from 1.567 min,1 (for the native enzyme) to 0.437 min,1 (for the stabilized enzyme). Similarly, the best crosslinking treatment increased the activation energy for denaturation from 391 kJ mol,1 (for the native protein) to 466 kJ mol,1 (for the stabilized enzyme). Crosslinking was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Enzyme-Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as Unique BiocatalystsBIOTECHNOLOGY PROGRESS, Issue 5 2002Hongfei Jia Improvement of catalytic efficiency of immobilized enzymes via materials engineering was demonstrated through the preparation of bioactive nanofibers. Bioactive polystyrene (PS) nanofibers with a typical diameter of 120 nm were prepared and examined for catalytic efficiency for biotransformations. The nanofibers were produced by electrospinning functionalized PS, followed by the chemical attachment of a model enzyme, ,-chymotrypsin. The observed enzyme loading as determined by active site titration was up to 1.4% (wt/wt), corresponding to over 27.4% monolayer coverage of the external surface of nanofibers. The apparent hydrolytic activity of the nanofibrous enzyme in aqueous solutions was over 65% of that of the native enzyme, indicating a high catalytic efficiency as compared to other forms of immobilized enzymes. Furthermore, nanofibrous ,-chymotrypsin exhibited a much-improved nonaqueous activity that was over 3 orders of magnitude higher than that of its native counterpart suspended in organic solvents including hexane and isooctane. It appeared that the covalent binding also improved the enzyme's stability against structural denaturation, such that the half-life of the nanofibrous enzyme in methanol was 18-fold longer than that of the native enzyme. [source] Monoclinic crystal form of Aspergillus niger,-amylase in complex with maltose at 1.8,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2006A. Vuji Aspergillus niger,-amylase catalyses the hydrolysis of ,-1,4-glucosidic bonds in starch. It shows 100% sequence identity to the A. oryzae homologue (also called TAKA-amylase), three crystal structures of which have been published to date. Two of them belong to the orthorhombic space group P212121 with one molecule per asymmetric unit and one belongs to the monoclinic space group P21 with three molecules per asymmetric unit. Here, the purification, crystallization and structure determination of A. niger,-amylase crystallized in the monoclinic space group P21 with two molecules per asymmetric unit in complex with maltose at 1.8,Å resolution is reported. Furthermore, a novel 1.6,Å resolution orthorhombic crystal form (space group P21212) of the native enzyme is presented. Four maltose molecules are observed in the maltose,,-amylase complex. Three of these occupy active-site subsites ,2 and ,1, +1 and +2 and the hitherto unobserved subsites +4 (Asp233, Gly234) and +5 (Asp235). The fourth maltose molecule binds at the distant binding sites d1 (Tyr382) and d2 (Trp385), also previously unobserved. Furthermore, it is shown that the active-site groove permits different binding modes of sugar units at subsites +1 and +2. This flexibility of the active-site cleft close to the catalytic centre might be needed for a productive binding of substrate chains and/or release of products. [source] New algorithms and an in silico benchmark for computational enzyme designPROTEIN SCIENCE, Issue 12 2006Alexandre Zanghellini Abstract The creation of novel enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Here we describe two new algorithms for enzyme design that employ hashing techniques to allow searching through large numbers of protein scaffolds for optimal catalytic site placement. We also describe an in silico benchmark, based on the recapitulation of the active sites of native enzymes, that allows rapid evaluation and testing of enzyme design methodologies. In the benchmark test, which consists of designing sites for each of 10 different chemical reactions in backbone scaffolds derived from 10 enzymes catalyzing the reactions, the new methods succeed in identifying the native site in the native scaffold and ranking it within the top five designs for six of the 10 reactions. The new methods can be directly applied to the design of new enzymes, and the benchmark provides a powerful in silico test for guiding improvements in computational enzyme design. [source] The effect of heavy atoms on the conformation of the active-site polypeptide loop in human ABO(H) blood-group glycosyltransferase BACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2007James A. Letts The human ABO(H) blood-group antigens are oligosaccharide structures that are expressed on erythrocyte and other cell surfaces. The terminal carbohydrate residue differs between the blood types and determines the immune reactivity of this antigen. Individuals with blood type A have a terminal N -acetylgalactosamine residue and those with blood type B have a terminal galactose residue. The attachment of these terminal carbohydrates are catalyzed by two different glycosyltransferases: an ,(1,3)N -acetylgalactosaminyltransferase (GTA) and an ,(1,3)galactosyltransferase (GTB) for blood types A and B, respectively. GTA and GTB are homologous enzymes that differ in only four of 354 amino-acid residues (Arg/Gly176, Gly/Ser235, Leu/Met266 and Gly/Ala268 in GTA and GTB, respectively). Diffraction-quality crystals of GTA and GTB have previously been grown from as little as 10,mg,ml,1 stock solutions in the presence of Hg, while diffraction-quality crystals of the native enzymes require much higher concentrations of protein. The structure of a single mutant C209A has been determined in the presence and absence of heavy atoms and reveals that when mercury is complexed with Cys209 it forces a significant level of disorder in a polypeptide loop (amino acids 179,195) that is known to cover the active site of the enzyme. The observation that the more highly disordered structure is more amenable to crystallization is surprising and the derivative provides insight into the mobility of this polypeptide loop compared with homologous enzymes. [source] | ||||||||||||||||