Molecular modelling and site-directed mutagenesis provide insight into saccharide pyruvylation by the Paenibacillus alvei CsaB enzyme.

Autor: Stefanović C; NanoGlycobiology Research Group, Department of Chemistry, Institute of Biochemistry, Universität für Bodenkultur Wien, Muthgasse 18, 1190, Vienna, Austria.; Department of Bionanosciences, Institute of Biologically Inspired Materials, Universität für Bodenkultur Wien, Muthgasse 11, 1190, Vienna, Austria., Hager-Mair FF; NanoGlycobiology Research Group, Department of Chemistry, Institute of Biochemistry, Universität für Bodenkultur Wien, Muthgasse 18, 1190, Vienna, Austria.; Department of Bionanosciences, Institute of Biologically Inspired Materials, Universität für Bodenkultur Wien, Muthgasse 11, 1190, Vienna, Austria., Breslmayr E; Department of Food Science and Technology, Biocatalysis and Biosensing Laboratory, Universität für Bodenkultur Wien, Muthgasse 11, 1190, Vienna, Austria.; Department of Material Sciences and Process Engineering, Institute for Molecular Modelling and Simulation, Universität für Bodenkultur Wien, Muthgasse 18, 1190, Vienna, Austria., López-Guzmán A; NanoGlycobiology Research Group, Department of Chemistry, Institute of Biochemistry, Universität für Bodenkultur Wien, Muthgasse 18, 1190, Vienna, Austria.; Department of Bionanosciences, Institute of Biologically Inspired Materials, Universität für Bodenkultur Wien, Muthgasse 11, 1190, Vienna, Austria.; Covirabio GmbH, Brehmstrasse 14a, 1110, Vienna, Austria., Lim C; Department of Chemistry, Institute of Organic Chemistry, Universität für Bodenkultur Wien, Muthgasse 18, 1190, Vienna, Austria., Blaukopf M; Department of Chemistry, Institute of Organic Chemistry, Universität für Bodenkultur Wien, Muthgasse 18, 1190, Vienna, Austria., Kosma P; Department of Chemistry, Institute of Organic Chemistry, Universität für Bodenkultur Wien, Muthgasse 18, 1190, Vienna, Austria., Oostenbrink C; Department of Material Sciences and Process Engineering, Institute for Molecular Modelling and Simulation, Universität für Bodenkultur Wien, Muthgasse 18, 1190, Vienna, Austria., Ludwig R; Department of Food Science and Technology, Biocatalysis and Biosensing Laboratory, Universität für Bodenkultur Wien, Muthgasse 11, 1190, Vienna, Austria., Schäffer C; NanoGlycobiology Research Group, Department of Chemistry, Institute of Biochemistry, Universität für Bodenkultur Wien, Muthgasse 18, 1190, Vienna, Austria. christina.schaeffer@boku.ac.at.; Department of Bionanosciences, Institute of Biologically Inspired Materials, Universität für Bodenkultur Wien, Muthgasse 11, 1190, Vienna, Austria. christina.schaeffer@boku.ac.at.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2023 Aug 17; Vol. 13 (1), pp. 13394. Date of Electronic Publication: 2023 Aug 17.
DOI: 10.1038/s41598-023-40072-1
Abstrakt: Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic β-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and K M and k cat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.
(© 2023. Springer Nature Limited.)
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje