Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme.This residue was therefore substituted in the mutant MeHNL-W128A by alanine to study its importance for the substrate specificity of the enzyme.Wild-type MeHNL and MeHNL-W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively).However, the specific activities of MeHNL-W128A for the unnatural substrates mandelonitrile and 4-hydroxymandelonitrile are increased 9-fold and ∼450-fold, respectively, compared with the wild-type MeHNL.The crystal structure of the MeHNL-W128A substratefree form at 2.1 Å resolution indicates that the W128A substitution has significantly enlarged the active-site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates.Surprisingly, the MeHNL-W128A-4-hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.