A7 Structural biology of glycosyltransferases
for the optimisation of biotechnical processes
Aim of the subproject A7 is the x-ray
analysis of a dextransucrase (including mutants) and a fructosyltransferase
to gain a detailed knowledge of the active centres of these enzymes
in sugar-free and sugar-bound form. The structures will allow for
a rational mutagenesis of the genes to evocate changes in substrate
specificities (subproject A3, Buchholz-Hofer).
An equally important aim is to design changes of the overall surface
charge of the proteins. In cooperation with subproject C2
(Seidel-Morgenstern) an improvement of separation processes by means
of mathematical simulation studies as well as the improved immobilisation
of enzymes (subproject C3 Buchholz-Jördening)
should be accomplished.
The program envisages the isolation, purification and crystallization
of a native dextransucrase followed by the x-ray structural analysis.
For that purpose, glycosyltransferases (GTF) from various organisms
will be used. Using expression in E. coli, GTF from S. oralis, strep-tag
fusioned gtfD from S. mutans as well as fructosyltransferase from
S. oralis will be produced.
Using expression in B. megaterium, DsrS of L. mesenteroides
will be produced. The subsequent crystallization of the cleaned
and functionally active enzymes will be employed by using commercially
available sparse-matrix screens.
In parallel mechanism-based inhibitors
such as 6-deoxysaccharose are being synthesized to support crystallization.
The structural data of these enzym-substrate complexes will allow
for the identification of amino acids vital for substrate recognition
and catalysis. The modelling of downstream processes for chromatography
based on interaction of protein surfaces and column materials is
supposed to be acquired in close cooperation with Teilprojekt C2
(Seidel-Morgenstern). In summary an optimization of biocatalysis
as well as the processing of enzymes for biocatalytic processes
will be reached.