Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

Protein Sci. 2002 Jan;11(1):65-71. doi: 10.1110/ps.33702.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde-Lyases / chemistry*
  • Binding Sites
  • Crystallography, X-Ray
  • Hydrogen / chemistry
  • Kinetics
  • Manihot / enzymology*
  • Models, Chemical
  • Models, Molecular
  • Mutation
  • Protein Binding
  • Tryptophan / chemistry*


  • Hydrogen
  • Tryptophan
  • Aldehyde-Lyases
  • hydroxymandelonitrile lyase

Associated data

  • PDB/1EB8
  • PDB/1EB9