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 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.
Lauble
Hanspeter
H
Institut für Organische Chemie der Universität Stuttgart, D-70569 Stuttgart, Germany. PeterLauble@t-online.de
Miehlich
Burkhard
B
Förster
Siegfried
S
Kobler
Christoph
C
Wajant
Harald
H
Effenberger
Franz
F
D000446:Aldehyde-Lyases / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D002365:Manihot / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D014364:Tryptophan / Q000737:chemistry*
D006859:Hydrogen; D014364:Tryptophan; D000446:Aldehyde-Lyases; C079359:hydroxymandelonitrile lyase