Characterization and Reshaping of a Large and Hydrophobic Nucleophile Pocket in Lipases/Acyltransferases
Résumé
Lipases/acyltransferases, such as CpLIP2 from Candida parapsilosis and CduLAc from Candida dubliniensis catalyze preferentially acyltransfer over hydrolysis when a suitable nucleophile is present, even in medium with a high thermodynamic activity of water (aW). They are related to CAL‐A from Moesziomyces antarcticus, which in comparison displays a lower acyltransfer ability. The 3D structures of wild‐types and mutants of CAL‐A, CpLIP2 and CduLAc revealed differences in size and hydrophobicity of a large pocket located under the catalytic triad. The kinetic behavior of site‐directed mutants confirmed the role of this pocket in the competition between methanol and water as the nucleophile acceptor for the deacylation step. The mutations provided a better understanding of key structural determinants for the variable levels of acyltransferase ability observed and supported the existence of a complex network of nucleophile interactions within the enzymes. The shape and size of the possible nucleophile pocket identified also suggested that multiple binding sites could exist, supporting the hypothesis of non‐overlapping leaving and accepting nucleophiles binding sites.