Towards 3rd generation scaffolds: Enzyme design for in vitro production of natural and functionalized spolySia
Zusammenfassung der Projektergebnisse
The aim of WP 2.2 was to generate polySTs applying in vitro evolution approaches that exhibit improved solubility and stability and altered substrate specificities. To identify and select enzymes with the desired characteristics, at first, high throughput screening systems were established as tools to cope with the high number of enzyme variants generated by in vitro evolution. A temperature controlled colony culture and blotting technique was developed that allowed the rapid and reliable detection of clones expressing polySia. Complementary, a polyST activity assay was established using DMB-labelled acceptors and detection via fluorescence and HPLC methods. Using this method, polySTs could be characterized by the form of their reaction time course (by taking samples at different time points and determining the number of Sia transfers) and the respective distribution of corresponding products, and polyST activity could even be quantified. As the synthesis of uniform glycan structures is of utmost importance for their use as therapeutic reagents, we did not only engineer enzyme stability and solubility, but simultaneously searched for enzymes with high distributive elongation mechanisms. These enzymes are easy to control by the simple relation of acceptor:donor. The results obtained in work package 2.2 significantly contribute to the basic understanding of structure and catalytic mechanism of bacterial polySTs and, thus, are essential for the design of tailor-made enzymes for the production of functionalized polySia - studies that are still in progress in projects succeeding FOR 548.
Projektbezogene Publikationen (Auswahl)
- (2010). Crystal structure of an intramolecular chaperone mediating triple-beta-helix folding. Nat. Struct. Mol. Biol., 17, 210-215
Schulz, E.C., Dickmanns, A., Urlaub, H., Schmitt, A., Mühlenhoff, M., Stummeyer, K., Schwarzer, D., Gerardy-Schahn, R., and Ficner, R.
(Siehe online unter https://doi.org/10.1038/nsmb.1746) - (2010). Neisseria meningitidis serogroup B polysialyltransferase: insights into substrate binding. Chembiochem., 11, 170-174
Böhm, R., Freiberger, F., Stummeyer, K., Gerardy-Schahn, R., von Itzstein, M., and Haselhorst, T.
(Siehe online unter https://doi.org/10.1002/cbic.200900659) - 2010). Structural basis for the recognition and cleavage of polysialic acid by the bacteriophage K1F tailspike protein EndoNF. J. Mol. Biol., 397, 341-351
Schulz, E.C., Schwarzer, D., Frank, M., Stummeyer, K., Mühlenhoff, M., Dickmanns, A., Gerardy-Schahn, R., and Ficner, R.
(Siehe online unter https://doi.org/10.1016/j.jmb.2010.01.028) - (2012). A high-throughput screen for polysialyltransferase activity. Anal. Biochem., 427, 60-68
Keys, T.G., Berger, M., and Gerardy-Schahn, R.
(Siehe online unter https://doi.org/10.1016/j.ab.2012.04.033) - (2012). A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases. Anal. Biochem., 427, 107-115
Keys, T.G., Freiberger, F., Ehrit, J., Krueger, J., Eggers, K., Buettner, F.F., and Gerardy-Schahn, R.
(Siehe online unter https://doi.org/10.1016/j.ab.2012.05.011) - (2012). Glycomic strategy for efficient linkage analysis of di-, oligo- and polysialic acids. J. Proteomics., 75, 5266-5278
Galuska, S.P., Geyer, H., Mink, W., Kaese, P., Kuhnhardt, S., Schafer, B., Mühlenhoff, M., Freiberger, F., Gerardy-Schahn, R., and Geyer, R.
(Siehe online unter https://doi.org/10.1016/j.jprot.2012.06.011) - (2013) Defining a Substrate Binding Model of a Polysialyltransferase. Chembiochem., 14(15):1949-53
Freiberger, F., Böhm, R., Schwarzer, D., Gerardy-Schahn, R., Haselhorst, T., and von Itzstein, M.
(Siehe online unter https://doi.org/10.1002/cbic.201300367) - (2013). A single amino acid toggles Escherichia coli polysialyltransferases between mono- and bifunctionality. Glycobiology, 23, 613-618
Keys, T.G., Fuchs, H.L., Galuska, S.P., Gerardy-Schahn, R., and Freiberger, F.
(Siehe online unter https://doi.org/10.1093/glycob/cwt003) - (2014) Engineering the product profile of a polysialyltransferase. Nat. Chem. Biol., 10, 437–442
Keys, T.G., Fuchs, L.S., Ehrit, J., Freiberger, F., and Gerardy-Schahn, R.
(Siehe online unter https://doi.org/10.1038/nchembio.1501)
