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.Keywords: Substrate specificity; active-site tunnel mutant; crystal structure Hydroxynitrile lyases (HNLs) constitute a diverse family of enzymes that catalyze the stereospecific cleavage of a wide range of cyanohydrins into aldehydes or ketones and hydrogen cyanide (cyanogenesis) , which have recently become the subject of intensive structural studies (Wagner et al. 1996;Zuegg et al. 1999; Lauble et al. 2001a,b). Apart from the interesting aspects of their catalytic function, HNLs are of practical importance as biocatalysts for the reverse reaction of cyanogenesis, that is, the stereoselective addition of HCN to carbonyl compounds (Scheme 1).Although the HNL from Manihot esculenta (MeHNL) accepts a broad range of carbonyl compounds as substrates , MeHNL-catalyzed cyanohydrin formation is limited by bulky substituents with respect to conversion and ee-value, as shown, for example, for 3-phenoxybenzaldehyde, an important starting material for pyrethroids Bühler 2000).On the basis of the X-ray structure of MeHNL (Lauble et al. 1999), first attempts have been undertaken to correlate the substrate specificity with structural features of the threedimensional structure of the enzyme. The X-ray structure of