|929-1||An insight into thermal stability of a xylanase: the influence of glycosylation analyzed by experimental results and molecular dynamics simulations|
|Autores:||Raquel Fonseca Maldonado (FMRP-USP - Faculdade de Medicina de Ribeirão Preto) ; Davi Serradella Vieira (FFCLRP-USP - Faculdade de Filosofia Ciências e Letras de Ribeirão Preto) ; Juliana Sanches Alponti (FFCLRP-USP - Faculdade de Filosofia Ciências e Letras de Ribeirão Preto) ; Eric Boneli (UDM - Université de Montréal) ; Pierre Thibault (IRIC - 3Institute for Research in Immunology and Cancer) ; Richard Jonh Ward (FMRP-USP - Faculdade de Medicina de Ribeirão Preto / FFCLRP-USP - Faculdade de Filosofia Ciências e Letras de Ribeirão Preto) |
The relationship between enzyme stability and glycosylation in the GH11 xylanase A from Bacillus subtilis (XylA, a 1,4-β-O xylanohydrolase) has been studied using heterologous protein produced in Pichia pastoris (XylAPp). Intact mass analyses before and after endoH digestion indicate 4 predominant GlcNAc2Man9 glycosylation events in the XylAPp. LC-MS/MS analyses of the XylAPp chymotrypsin digestion products support glycosylation at N20, N25, N141 and N181. Single and double site-directed mutants that eliminated the N-glycosylation sites revealed that in mutants N20Q, N25Q, and N181Q, the N29 (which is not modified in wild-type XylAPpp) is glycosylated.A substantially higher thermotolerance of the XylAPpp and some mutants was observed in the glycosylated enzymes, where at 55oC the half-life of the glycosylated XylAPpp was 24 min, as compared to 8 min for the unglycosylated XylAEcp. Stability is reduced by 90% in the N20Q mutant, yet improved in the N25Q and N181Q mutants. The half-life of the N25Q/N181Q mutant was over 23 hours and LC-MS/MS analyses indicated glycosylations at N20, N29 and N141. These results were correlated with molecular dynamics simulations, which suggested that sugar-protein contacts contribute to increase the stability of the protein. Stabilization are monitorized by root mean square deviatin (RMSD) along the MD simulation, calculated for the backbone atoms of the protein. The glycan at N20 position present a high capacity of coverage of the protein surface, that is, it maximizes protein-glycan interactions. This coverage by glycan at N20 position ocurrs significantly when isolated or combined with other positions, but not whatever positions. The glycosylated N181 position decrease such functionality of glycosylated N20 position by direct glycan-glycan interaction. The results presented illustrate that the position of glycosylation may be far more important for protein thermostability than the amount of glycan added. In a general view, the MD simulation results shows the glycosylated positions define which regions of the protein surface are covered, modulate the glycan-glycan interactions which indirectly also defines the protein|glycan interface, define how efficiently the glycans interact to the protein surface and how the combination of glycosylated positions can contribute to improving or disturbing the protein|glycan interface.
Palavras-chave: xylanase, N-glycosylation, protein stability, simulation, mass spectometry