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Structure-function relationships of glucosyltransferases

Ziel

The objective of the work is to elucidate and to understand at the molecular level the mechanism and action of glucosyl-transferases. Two enzymes, cyclodextringlucosyltransferase (CGTase) and dextransucrase will be studied and their mechanism of action and acceptor specificity compared at the molecular level. An improved understanding of the mechanism of action will allow an improved control of the specificity and regioselectivity of the enzymes.
CGTase:
The Bacillus circulans wild type CGTase was cloned and sequenced. Suitable vectors were developed which allowed site-directed mutagenesis and overproduction of the CGTase protein. Purified CGTase was crystallized and the 3D structure determined. Soaking studies with crystals of the CGTase protein and the inhibitor acarbose and/or oligosaccharides and/or cyclodextrins followed by X-ray diffraction studies resulted in the elucidation of 3D structures of CGTase protein molecules containing oligosaccharides of various lengths, bound in the active site cleft or in the raw starch binding domain. This has allowed identification amino acid residues interacting with sugar substrates and products. This information provided clear insight in factors determining CGTase cyclodextrin product selectivity and product inhibition. On the basis of this data clear suggestions for targets for site-directed mutagenesis became available, allowing protein engineering of CGTase to construct mutant proteins with improved cyclodextrin product specificity and reduced product inhibition. This was a major breakthrough, allowing rational construction of improved variants of commercial thermostable CGTase proteins.
A detailed CGTase sequence analysis with identification of important residues has been carried out.
The kinetic behaviour of CGTase has been studied with the aim of obtaining an improved understanding of the reaction mecha-nisms of glucosyltransferases. Because of the low specificity of CGTase with respect to oligosaccharide substrates and the heterogenicity of the products the development of a closed model has been a difficult task. A first model is now in use for the simulation of the experiments.
The stability of CGTase from Thermoanaerobacter (T-CGTase) in the absence of substrate was determined in organic solvents from 0-100% solvent concentration. Residual hydrolysis activity was measured after 24 hours incubation at 25(C. T-CGTase retained over 70% hydrolysis activity (relative to enzyme incubated in the absence of solvent) in a wide range of polar solvents. The T-CGTase was only significantly inactivated in DMSO, DMF, and DMA at over 70% organic solvent concentration. There was no correlation between enzyme stability and hydrophobic properties of the solvents as determined by logP and dielectric constant. The results indicate the robust nature of this strain of CGTase.
Chemical modification of carboxyl groups of CGTase was performed with a water soluble carbodiimide, EDC, and an appropriate nucleophile (either glycine ethyl ester or norleucine methyl ester).
Dextransucrase:
A dextransucrase encoding gene (dsrA) from Leuconostoc mesenteroides NRRL B-1299 was isolated. The aminoacids exhibited a high similarity with other glucosyltransferases (GTF). The two domains already described in GTFs are conserved in DSRA: a N-terminal conserved domain and a C-terminal domain composed of series of repeats. Surprisingly, the expected signal peptide was not detected. The dextran produced appeared to be composed of 85% alpha(1-6) and 15% alpha(1-3) linkages and the oligosaccharides synthesised in the presence of maltose were mainly composed of alpha(1-6) linkages.
As DSRA was not the dextransucrase from L. mesenteroides NRRL B-1299 producing alpha(1-2) linkages, construction of mutants were carried out with dextransucrase from L. mesenteroides NRRL B-512F (DSR-S).
Aminoacid sequence comparisons between DSR-S and other GTFs revealed four conserved residues, D-511, D-513, D-551 and H-661. They were replaced by N-511, N-513, N-551 and R-661. While mutations at D-513 and H-661 significantly reduced the enzymatic activity of the mutant enzymes, replacements at D-511 and D-551 completely abolished the two enzymatic activities of the corresponding dextransucrases. However, glucan binding properties were retained in all mutants. That showed that at least two carboxyl groups (D-511 and D-551) are necessary for catalysis and that DSR-S, like other GTFs, has a two domain structure.
A limited model of the dextransucrase catalytic domain based on CGTase structures has been obtained.
Since dextransucrase acceptor reactions offer a new perspective for carbohydrate synthesis the correlation between the chemical structure of carbohydrates and the corresponding acceptor properties has been studied. From a broad range of studied potential acceptors we got a lot of informations concerning the influence of the several positions at acceptor-glucosyl-residue on the change of activity. In this context we developed a model for the complex kinetics of the dextransucrase, getting a useful tool for both comparing the activity of the enzyme to several reactor configuration for the production of acceptor products.
The kinetics of the dextransucrase reaction using maltose as acceptor was studied in the presence of a wide range of organic solvents. The highest activity was obtained with DMSO as cosolvent.
Covalent immobilization of the dextran free enzyme on silica beads has been carried out. The procedure is based on the coupling between the amino groups of the enzyme and the aldehyde moiety of the silica beads activated with glutaraldehyde.
MAJOR SCIENTIFIC BREAKTHROUGHS:
Increasing insights in the mechanisms of the CGTase responsible for product specificity and product inhibition has made construction possible(via site directed mutagenesis) of CGTase-variants with a more specific cyclodextrin production.
A novel enzyme from L. mesenteroides NRRL B-1299 synthesizing a polymer consisting of 90% alpha(1-6)-linkages and 10% alpha(1-3)-linkages has been found.
New potential products from the reaction of dextransucrase with unusual acceptors have been identified.
A limited model of the dextransucrase catalytic domain based on CGTase structures has been obtained.

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Letzte Aktualisierung: 31 Dezember 1997

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Permalink: https://cordis.europa.eu/project/id/BIO2943071/de

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