Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

Abstract

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 approximately 450-fold, respectively, compared with the wild-type MeHNL. The crystal structure of the MeHNL-W128A substrate-free form at 2.1 A 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 A 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.

Fig. 1.

(A) Molecular surface drawing of wild-type MeHNL (left) and MeHNL-W128A-4–hydroxybenzaldehyde complex (right). The enzymes are viewed toward the active-site tunnel entrance. The surface of Trp 128 (left) and Ala 128 (right) is colored yellow, showing the completely covered entrance of the wild-type enzyme and the wide-open substrate binding site of the mutant enzyme. Stereoview of the MeHNL-W128A active site in complex with 4-hydroxybenzaldehyde. (B) |F_o|-|F_c| electron density omit map contoured at 4σ. (C) Intermolecular interactions of the substrate molecules and selected active-site residues. Hydrogen bonds are shown as red broken lines, van der Waals interactions as black broken lines.

Scheme 1.