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Donor Substrate (donor + substrate)
Selected AbstractsIdentification of catalytically important residues in the active site of Escherichia coli transaldolaseFEBS JOURNAL, Issue 8 2001Ulrich Schörken The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB -deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon,carbon bond cleavage and stabilization of the carbanion/enamine intermediate. [source] A Mutant D -Fructose-6-Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone for the Synthesis of Polyhydroxylated CompoundsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6 2010Abstract A mutant of D -fructose-6-phosphate aldolase (FSA) of Escherichia coli, FSA A129S, with improved catalytic efficiency towards dihydroxyacetone (DHA), the donor substrate in aldol addition reactions, was explored for synthetic applications. The kcat/KM value for DHA was 17-fold higher with FSA A129S than that with FSA wild type (FSA wt). On the other hand, for hydroxyacetone as donor substrate FSA A129S was found to be 3.5-fold less efficient than FSA wt. Furthermore, FSA A129S also accepted glycolaldehyde (GA) as donor substrate with 3.3-fold lower affinity than FSA wt. This differential selectivity of both FSA wt and FSA A129S for GA makes them complementary biocatalysts allowing a control over donor and acceptor roles, which is particularly useful in carboligation multi-step cascade synthesis of polyhydroxylated complex compounds. Production of the mutant protein was also improved for its convenient use in synthesis. Several carbohydrates and nitrocyclitols were efficiently prepared, demonstrating the versatile potential of FSA A129S as biocatalyst in organic synthesis. [source] Glycosyltransferase Microarray Displayed on the Glycolipid LB MembraneADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6-7 2003Noriko Nagahori Abstract ,(1,4),Galactosyltransferase expressed as a fusion protein with maltose binding protein (MBP-GalT) was displayed specifically on a Langmuir,Blodgett (LB) membrane prepared by photopolymerization of maltotriose-carrying glycolipid (1) with 1,2-bis(10,12-tricosadiynoyl)- sn -glycero-3-phosphocholine (2). The catalytic activity of MBP-GalT on the LB film was directly monitored by the surface plasmon resonance (SPR) method using a GlcNAc-carrying water-soluble polymer (3) as an acceptor substrate. Highly sensitive sigmoidal-type signals were obtained upon the addition of the acceptor substrate in the presence of the donor substrate, UDP-galactose (UDP-Gal), while the binding of 3 was not detected in the absence of UDP-Gal. The intensities of the signals were dependent on the amount of immobilized MBP-GalT on the LB film, which was estimated from the images obtained by atomic force microscope (AFM). [source] FRET-Based Direct and Continuous Monitoring of Human Fucosyltransferases Activity: An Efficient synthesis of Versatile GDP- L -Fucose Derivatives from Abundant d- Galactose,CHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2008Takahiro Maeda Abstract We have developed a facile and versatile protocol for the continuous monitoring of human fucosyltransferases activity by using fluorescence energy resonance transfer (FRET), and have explored the feasibility of its use in an inhibitor screening assay. A convenient sugar nucleotide with a fluorogenic probe, 6-deoxy-6- N -(2-naphalene-2-yl-acetamide)-,- L -galactopyranos-1-yl-guanosine 5,-diphosphate disodium salt (1), was efficiently synthesized from naturally abundant D -galactopyranose via a key intermediate, 6-azide-1,2,3,4-tetra- O -benzoyl-6-deoxy-,- L -galactopyranose (10). It was demonstrated that the combined use of the glycosyl donor 1 and a dansylated acceptor substrate, sialyl-,2,3-LacNAc derivative (2) allowed us to carry out highly sensitive, direct, and continuous in vitro monitoring of the generation of sialyl Lewis,X (SLex), which is catalyzed by human ,-1,3-fucosyltransferase,VI (FUT-VI). A kinetic analysis revealed that compound 1 was an excellent donor substrate (KM=0.94,,M and Vmax=0.14,,M,min,1) for detecting human FUT-VI activity. To the best of our knowledge, this synthetic fluorogenic probe is the most sensitive and selective donor substrate for FUT-VI among all of the known GDP-Fuc analogues, including the parent GDP-Fuc. When a dansylated asparagine-linked glycopeptide 20, which is derived from egg yolk was employed as an alternate acceptor substrate, a FRET-based assay with compound 1 could be used to directly monitor the ,1,6-fucosylation at the reducing terminal GlcNAc residue by human FUT-VIII (KM=175,,M and Vmax=0.06,,M/,min); this indicates that the present method might become a general protocol for the characterization of various mammalian fucosyltransferases in the presence of designated fluorogenic acceptor substrates. The present protocol revealed that compound 23, which was obtained by a 1,3-dipolar cycloaddition between the disodium salt 16 and 1-ethynyl-naphthalene exhibits highly potent inhibitory effects against the FUT-VI-mediated sialyl Lewis,X synthesis (IC50=5.4,,M). [source] Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthodermaFEBS JOURNAL, Issue 3 2007Magdalena Kujawa We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose,methanol,choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9- S -cysteinyl, 8-,- N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are d -glucose, d -galactose, l -arabinose, and d -xylose. As shown by in situ NMR analysis, d -glucose and d -galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (kcat/Km). The enzyme may play a role in lignocellulose degradation. [source] | ||||||||||