Molecular dynamics simulations have been performed on the lipase B from Candida antarctica (CalB) in its native form and with one or two oxidised residues, either methionine oxidised to methionine sulphoxide, tryptophan oxidised to 5-hydroxytryptophan, or cystine oxidised to a pair of cysteic acid residues. We have analysed how these mutations affect the general structure of the protein, as well as the local structure around the oxidised amino acid and the active site. The results indicate that the methionine and tryptophan oxidations led to rather restricted changes in the structure, whereas the oxidation of cystines, which also caused cleavage of the cystine S–S linkage, gave rise to larger changes in the protein structure. Only two oxidised residues caused significant changes in the structure of the active site, viz. those of the Cys-22/64 and Cys-216/258 pairs. Site-directed mutagenesis studies were also performed. Two variants showed a similar behaviour as native CalB (M83I and M129L), whereas W155Q and M72S had severely decreased specific activity. M83I had a slightly higher thermostability than native CalB. No significant increase in stability towards hydrogen peroxide was observed. The same mutants were also studied by molecular dynamics. Even though no significant increase in stability towards hydrogen peroxide was observed, the results from simulations and site-directed mutagenesis give some clues to the direction of further work on stabilization.
