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Active Enzyme (active + enzyme)
Selected AbstractsChemoenzymatic Synthesis of Branched Oligo- and Polysaccharides as Potential Substrates for Starch Active Enzymes.CHEMINFORM, Issue 9 2004Lionel Greffe No abstract is available for this article. [source] The two N-glycans present on bovine Pofut1 are differently involved in its solubility and activityFEBS JOURNAL, Issue 5 2007Céline Loriol O-Fucosylation is a post-translational glycosylation in which an O -fucose is covalently attached to the hydroxyl group of a specific serine or threonine residue. This modification occurs within the consensus sequence C2X4,5(S/T)C3 present on epidermal growth factor-like repeats of several proteins, including the Notch receptors and their ligands. The enzyme responsible for the addition of O -fucose to epidermal growth factor-like repeats is protein O -fucosyltransferase 1. Protein O -fucosyltransferase 1-mediated O -fucosylation is essential in Notch signaling, folding and targeting to the cell surface. Here, we studied the expression pattern of protein O -fucosyltransferase 1 in cattle and showed that the active enzyme is present in all tissues examined from embryo and adult as a glycoprotein with two N-glycans. By comparing protein O -fucosyltransferase 1 sequences available in databases, we observed that mammalian protein O -fucosyltransferase 1 enzymes possess two putative N-glycosylation sites, and that only the first is conserved among bilaterians. To gain more insight regarding the significance of N-glycans on protein O -fucosyltransferase 1, we substituted, by site-directed mutagenesis, bovine protein O -fucosyltransferase 1 N65, N163 or both, with L or Q. We demonstrated that the loss of N-glycan on N163 caused a slight decrease in protein O -fucosyltransferase 1 activity. In contrast, glycosylation of N65 was crucial for protein O -fucosyltransferase 1 functionality. Loss of glycosylation at N65 resulted in aggregation of protein O -fucosyltransferase 1, suggesting that N-glycosylation at this site is essential for proper folding of the enzyme. [source] Kinetics of intra- and intermolecular zymogen activation with formation of an enzyme,zymogen complexFEBS JOURNAL, Issue 1 2005Matilde Esther Fuentes A mathematical description was made of an autocatalytic zymogen activation mechanism involving both intra- and intermolecular routes. The reversible formation of an active intermediary enzyme,zymogen complex was included in the intermolecular activation route, thus allowing a Michaelis,Menten constant to be defined for the activation of the zymogen towards the active enzyme. Time,concentration equations describing the evolution of the species involved in the system were obtained. In addition, we have derived the corresponding kinetic equations for particular cases of the general model studied. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. The validity of the results obtained were checked by using simulated progress curves of the species involved. The model is generally good enough to be applied to the experimental kinetic study of the activation of different zymogens of physiological interest. The system is illustrated by following the transformation kinetics of pepsinogen into pepsin. [source] Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experimentsFEBS JOURNAL, Issue 22 2001Sébastien Dementin Enzymes MurD, MurE, MurF, folylpolyglutamate synthetase and cyanophycin synthetase, which belong to the Mur synthetase superfamily, possess an invariant lysine residue (K198 in the Escherichia coli MurD numbering). Crystallographic analysis of MurD and MurE has recently shown that this residue is present as a carbamate derivative, a modification presumably essential for Mg2+ binding and acyl phosphate formation. In the present work, the importance of the carbamoylated residue was investigated in MurD, MurE and MurF by site-directed mutagenesis and chemical rescue experiments. Mutant proteins MurD K198A/F, MurE K224A and MurF K202A, which displayed low enzymatic activity, were rescued by incubation with short-chain carboxylic acids, but not amines. The best rescuing agent was acetate for MurD K198A, formate for K198F, and propionate for MurE K224A and MurF K202A. In the last of these, wild-type levels of activity were recovered. A complementarity between the volume of the residue replacing lysine and the length of the carbon chain of the acid was noted. These observations support a functional role for the carbamate in the three Mur synthetases. Experiments aimed at recovering an active enzyme by introducing an acidic residue in place of the invariant lysine residue were also undertaken. Mutant protein MurD K198E was weakly active and was rescued by formate, indicating the necessity of correct positioning of the acidic function with respect to the peptide backbone. Attempts at covalent rescue of mutant protein MurD K198C failed because of its lack of reactivity towards haloacids. [source] Engineering the properties of a cold active enzyme through rational redesign of the active siteFEBS JOURNAL, Issue 19 2001Iason Tsigos ,In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5,25 °C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5,15 °C and a significantly increased activity at 20,25 °C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135,Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints. [source] Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylaseFEBS JOURNAL, Issue 23 2000Evidence that GDP-mannose, GDP-Glc pyrophosphorylases are different proteins GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1- P to GTP to form GDP-Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an , subunit (43 kDa) and a , subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1- P and GTP) and GDP-Man (from Man-1- P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the , subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the , subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native , subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1- P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1- P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the , subunit is the GMPP, whereas the , subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity. [source] Analysis of the thyrotropin-releasing hormone-degrading ectoenzyme by site-directed mutagenesis of cysteine residuesFEBS JOURNAL, Issue 9 2000Cys68 is involved in disulfide-linked dimerization Thyrotropin-releasing hormone-degrading ectoenzyme is a member of the M1 family of Zn-dependent aminopeptidases and catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH-DE we used a site-directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein,protein interactions but not via interchain S-S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C-terminal domain remote from the Zn-binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane-spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface. [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] A novel aspartylglucosaminuria mutation affects translocation of aspartylglucosaminidase,,HUMAN MUTATION, Issue 4 2004Jani Saarela Abstract The AGA gene is mutated in patients with aspartylglucosaminuria (AGU), a lysosomal storage disease enriched in the Finnish population. The disease mechanism of AGU and the biochemistry and cell biology of the lysosomal aspartylglucosaminidase (AGA) enzyme are well characterized. Here, we have investigated a novel AGU mutation found in a Finnish patient. The mutation was detected as a compound heterozygote with the Finnish major mutation in the other allele. The novel point mutation, c.44T>G, causes the L15R amino acid substitution in the signal sequence of the AGA enzyme. The mutated AGA enzyme was here analyzed by over expression in BHK and COS-1 cells. The L15R AGA protein was only faintly detectable by immunofluorescence analysis and observed in the endoplasmic reticulum. Metabolic labeling and immunoprecipitation revealed only a small amount of AGA polypeptides but the specific activity of the mutant enzyme was surprisingly high, 37% of the wild type. The amino acid substitution probably affects translocation of AGA polypeptides by altering a critical hydrophobic core structure of the signal sequence. It appears that the small amounts of active enzyme are not able to reach the lysosomes thus explaining the development of AGU disease in the patient. © 2004 Wiley-Liss, Inc. [source] Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodiesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008D.E.C.S. Rao Abstract Aims:, To isolate, clone and express a novel phytase gene (phy) from Bacillus sp. in Escherichia coli; to recover the active enzyme from inclusion bodies; and to characterize the recombinant phytase. Methods and Results:, The molecular weight of phytase was estimated as 40 kDa on SDS-polyacrylamide gel electrophoresis. A requirement of Ca2+ ions was found essential both for refolding and activity of the enzyme. Bacillus phytase exhibited a specific activity of 16 U mg,1 protein; it also revealed broad pH and temperature ranges of 5·0 to 8·0 and 25 to 70°C, respectively. The Km value of phytase for hydrolysis of sodium phytate has been determined as 0·392 mmol l,1. The activity of enzyme has been inhibited by EDTA. The enzyme exhibited ample thermostability upon exposure to high temperatures from 75 to 95°C. After 9 h of cultivation of transformed E. coli in the bioreactor, the cell biomass reached 26·81 g wet weight (ww) per l accounting for 4289 U enzyme activity compared with 1·978 g ww per l producing 256 U activity in shake-flask cultures. In silico analysis revealed a ,-propeller structure of phytase. Conclusions:, This is the first report of its kind on the purification and successful in vitro refolding of Bacillus phytase from the inclusion bodies formed in the transformed E. coli. Significance and Impact of the Study:, Efficient and reproducible protocols for cloning, expression, purification and in vitro refolding of Bacillus phytase enzyme from the transformed E. coli have been developed. The novel phytase, with broad pH and temperature range, renaturation ability and substrate specificity, appears promising as an ideal feed supplement. Identification of site between 179th amino acid leucine and 180th amino acid asparagine offers scope for insertion of small peptides/domains for production of chimeric genes without altering enzyme activity. [source] Stromelysin-3 suppresses tumor cell apoptosis in a murine model,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2001Erxi Wu Abstract Stromelysin-3 (STR-3) is a matrix metalloproteinase with a unique pattern of expression and substrate specificity. During embryogenesis and remodeling of normal adult tissues, STR-3 is produced by stromal cells in direct contact with epithelial cells undergoing regional apoptosis and selective cell survival. STR-3 is also overexpressed by interdigitating stromal cells in primary epithelial malignancies. Although STR-3 does not degrade classic extracellular matrix components, the enzyme promotes the establishment of local tumors in nude mice by as yet undefined mechanisms. STR-3 is induced when malignant epithelial cells come into contact with surrounding stromal elements; the active stromal cell-derived 45 kDa enzyme is subsequently processed to a 35 kDa protein without enzymatic activity. We have generated MCF-7 transfectants expressing wild type or catalytically inactive 45 kDa STR-3 (STR-3wt and STR-3cat- ) or secreted 35 kDa STR-3 (35 kDa STR-3sec) and evaluated their implantation and survival in nude mice. Tumors developed significantly more rapidly in animals receiving STR-3wt, rather than vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Most importantly, STR-3wt tumors had a significantly lower percentage of apoptotic cells than tumors derived from vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Taken together, these studies suggest that the active STR-3 enzyme may increase tumor take by suppressing tumor cell apoptosis and that 45 kDa to 35 kDa STR-3 processing limits STR-3 activity at the tumor/stromal interface. Because STR-3 is secreted as an active enzyme rather than a proform, subsequent 45 kDa to 35 kDa STR-3 processing may represent a novel mechanism for regulating enzymatic activity. J. Cell. Biochem. 82: 549,555, 2001. © 2001 Wiley-Liss, Inc. [source] THERMORESISTANCE OF PECTIN METHYLESTERASE IN SANGUINELLO ORANGE JUICEJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2001FRANCESCO DE SIO ABSTRACT The behavior of pectin methylesterase (EC 3.1.1.11) activity as a function of temperature was investigated in Sanguinello orange juice. The thermal inactivation of this enzyme was evaluated in the range of 75,95C and was found to be nonlinear. Due to very low activity content, after the thermal treatment at temperatures above 80C, the active enzyme was concentrated by affinity chromatography. The logarithmic values of decimal reduction times, plotted against temperature, showed a nonlinear pattern featuring a sudden change in slope at temperatures exceeding 85C. The z value determined in the range 85,95C was much higher than that calculated in the range 75,85 C. The decimal reduction times were used to estimate the times of treatment for the enzyme inactivation during a HTST process. [source] Cell transformation induced by hepatitis C virus NS3 serine proteaseJOURNAL OF VIRAL HEPATITIS, Issue 2 2001R. Zemel Persistent infection with hepatitis C virus (HCV) may lead to hepatocellular carcinoma (HCC). It has been suggested that HCV-encoded proteins are directly involved in the tumorigenic process. The HCV nonstructural protein NS3 has been identified as a virus-encoded serine protease. To study whether HCV NS3 has oncogenic activity, nontumorigenic rat fibroblast (RF) cells were stably transfected with an expression vector containing cDNA for the NS3 serine protease (nucleotides 3356,4080). The NS3 serine protease activity was determined in the transfected cells. The transfected cells grew rapidly and proliferated serum independently, lost contact inhibition, grew anchorage independently in soft agar and induced significant tumour formation in nude mice. Cells transfected with an expression vector containing a mutated NS3 serine protease (serine 139 to alanine at the catalytic site) showed no transforming abilities; their growth was dependent on serum and they did not grow anchorage independently in soft agar. Moreover, cells transfected with the NS3 serine protease and treated with the chymotrypsin inhibitors TPCK and PMSF (a serine protease inhibitor) lost their transforming feature. These results suggest that the NS3 serine protease of HCV is involved in cell transformation and that the ability to transform requires an active enzyme. [source] Characterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase,thymidylate synthase from Plasmodium falciparumACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2004Penchit Chitnumsub The full-length pfdhfr-ts genes of the wild-type TM4/8.2 and the double mutant K1CB1 (C59R+S108N) from the genomic DNA of the corresponding Plasmodium falciparum parasite have been cloned into a modified pET(17b) plasmid and expressed in Escherichia coli BL21 (DE3) pLysS. Conditions for the expression and purification of the P. falciparum dihydrofolate reductase,thymidylate synthase (PfDHFR-TS) have been established that yield ,1,mg of the soluble active enzyme per litre of culture. The purified enzymes have been crystallized using a modified microbatch method with PEG 4000 as the primary precipitating agent. X-ray diffraction data were collected to 2.50 and 2.64,Å resolution under cryogenic conditions from single crystals of the two PfDHFR-TS proteins in complex with NADPH, dUMP and either Pyr30 or Pyr39. Preliminary X-ray analysis indicated that the crystals belong to the orthorhombic space group P212121, with two molecules per asymmetric unit and ,52% solvent content (VM, 2.6,Å3,Da,1). The use of a particular type of baby oil in the microbatch setup appeared to be beneficial to PfDHFR-TS crystallization and a preliminary comparison with another commonly used oil is described. [source] Crystallization and X-ray diffraction analysis of an all-RNA U39C mutant of the minimal hairpin ribozymeACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2003Valerie Grum-Tokars The hairpin ribozyme is a naturally occurring catalytic RNA composed of two helix,loop,helix domains, A and B, that dock to form the biologically active enzyme. Previously, the crystal structure of the hairpin has been solved as a four-way helical junction that incorporated the U1A protein as an artificial crystal-packing motif [Rupert & Ferré-D'Amaré (2001), Nature (London), 410, 780,786]. Here, the crystallization of a minimal junctionless hairpin ribozyme 64-mer is reported in the absence of protein. Crystals grow in space group P6122, with unit-cell parameters a = 93.1, c = 123.2,Å. Complete diffraction data have been collected to 3.35,Å resolution. Structural analysis should provide details of intermolecular RNA docking, including the ground-state conformations of the U39C mutation relevant to hairpin catalysis. [source] Crystallization and preliminary X-ray crystallographic studies of recombinant human betaine,homocysteine S-methyltransferaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2001Nandita Bose Betaine,homocysteine S-methyltransferase (BHMT) catalyzes a reaction essential for regulation of methionine and homocysteine metabolism and the catabolism of choline in mammalian tissues. Human recombinant BHMT (MW = 45,kDa) has been crystallized by the hanging-drop vapor-diffusion method at 294,K using ethylene glycol as the precipitant. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 109.190, b = 91.319, c = 88.661,Å, , = 122.044°, and diffract to 2.9,Å resolution on a local rotating-anode X-ray source. Rotation-function analysis and the Matthews coefficient, VM = 2.46,Å3,Da,1, are consistent with a dimer in the asymmetric unit, suggesting that the active enzyme is a tetramer with 222 symmetry. [source] Crystallization and preliminary X-ray crystallographic studies on the bacteriophage ,6 RNA-dependent RNA polymeraseACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2000Sarah J. Butcher The RNA-dependent RNA polymerase (P2) from bacteriophage ,6 has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme. A fully substituted selenomethionyl version of the protein has also been produced. Crystals of both proteins have been grown; most belong to the monoclinic space group P21, with unit-cell parameters a = 105.9, b = 94.0, c = 140.9,Å, , = 101.4°, but some are trigonal (space group P31 or P32), with unit-cell parameters a = b = 110.1, c = 159.4,Å, , = 120°. Both crystal forms occur in the same crystallization drop and are morphologically indistinguishable. Native data sets have been collected from both types of crystals to better than 3,Å resolution. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of chlorite dismutase: a detoxifying enzyme producing molecular oxygenACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2008Daniël C. De Geus Chlorite dismutase, a homotetrameric haem-based protein, is one of the key enzymes of (per)chlorate-reducing bacteria. It is highly active (>2,kU,mg,1) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction. The chlorite dismutase gene in Azospira oryzae strain GR-1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli. Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P21212 and were most likely to contain six subunits in the asymmetric unit. The refined unit-cell parameters were a = 164.46, b = 169.34, c = 60.79,Å. The crystals diffracted X-rays to 2.1,Å resolution on a synchrotron-radiation source and a three-wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available. [source] Redox and antioxidant systems of the malaria parasite Plasmodium falciparumMOLECULAR MICROBIOLOGY, Issue 5 2004Sylke Müller Summary The malaria parasite Plasmodium falciparum is highly adapted to cope with the oxidative stress to which it is exposed during the erythrocytic stages of its life cycle. This includes the defence against oxidative insults arising from the parasite's metabolism of haemoglobin which results in the formation of reactive oxygen species and the release of toxic ferriprotoporphyrin IX. Central to the parasite's defences are superoxide dismutases and thioredoxin-dependent peroxidases; however, they lack catalase and glutathione peroxidases. The vital importance of the thioredoxin redox cycle (comprising NADPH, thioredoxin reductase and thioredoxin) is emphasized by the confirmation that thioredoxin reductase is essential for the survival of intraerythrocytic P. falciparum. The parasites also contain a fully functional glutathione redox system and the low-molecular-weight thiol glutathione is not only an important intracellular thiol redox buffer but also a cofactor for several redox active enzymes such as glutathione S-transferase and glutaredoxin. Recent findings have shown that in addition to these cytosolic redox systems the parasite also has an important mitochondrial antioxidant defence system and it is suggested that lipoic acid plays a pivotal part in defending the organelle from oxidative damage. [source] Inhibitors of plant invertases do not affect the structurally related enzymes of fructan metabolismNEW PHYTOLOGIST, Issue 3 2009Ute Kusch Summary ,,Plant fructan active enzymes (FAZYs), including the enzymes involved in inulin metabolism, namely sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) and fructan 1-exohydrolase (1-FEH; EC 3.2.1.153), are evolutionarily related to acid invertases (AIs), that is, plant cell wall invertase (CWI) and vacuolar invertase (VI). Acid invertases are post-translationally controlled by proteinaceous inhibitors. Whether FAZYs are subject to similar controls is not known. ,,To probe their possible interactions with invertase inhibitors, we transiently expressed chicory (Cichorium intybus) FAZYs, as well as several previously characterized invertase inhibitors from nonfructan species, and the C. intybus cell wall/vacuolar inhibitor of fructosidase (CiC/VIF), a putative invertase inhibitor of a fructan-accumulating plant, in leaves of Nicotiana benthamiana. ,,Leaf extracts containing recombinant, enzymatically active FAZYs were used to explore the interaction with invertase inhibitors. Neither heterologous inhibitors nor CiC/VIF affected FAZY activities. CiC/VIF was confirmed as an AI inhibitor with a stronger effect on CWI than on VI. Its expression in planta was developmentally regulated (high in taproots, and undetectable in leaves and flowers). In agreement with its target specificities, CiC/VIF was associated with the cell wall. ,,It is concluded that subtle structural differences between AIs and FAZYs result in pronounced selectivity of inhibitor action. [source] TEM-1 ,-lactamase as a scaffold for protein recognition and assayPROTEIN SCIENCE, Issue 6 2002Daniel Legendre Abstract A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 ,-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to ,-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that ,-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to ,-lactamases binding to streptavidin, ,-lactamase clones binding to horse spleen ferritin and ,-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining ,-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for ,-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme. [source] | |||||||||||||