Enzyme-Catalyzed Reactions, Part 47. Part 46: J. Roos, F. Effenberger, Tetrahedron: Asymmetry 2002, 13, 1855–1862.
Substrate Specificity of Mutants of the Hydroxynitrile Lyase from Manihot esculenta†
Article first published online: 27 FEB 2003
Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Volume 4, Issue 2-3, pages 211–216, March 3, 2003
How to Cite
Bühler, H., Effenberger, F., Förster, S., Roos, J. and Wajant, H. (2003), Substrate Specificity of Mutants of the Hydroxynitrile Lyase from Manihot esculenta. ChemBioChem, 4: 211–216. doi: 10.1002/cbic.200390033
- Issue published online: 27 FEB 2003
- Article first published online: 27 FEB 2003
- Manuscript Received: 26 SEP 2002
- bioorganic chemistry;
Several tryptophan128-substituted mutants of the hydroxynitrile lyase from Manihot esculenta (MeHNL) are constructed and applied in the MeHNL-catalyzed addition of HCN to various aromatic and aliphatic aldehydes as well as to methyl and ethyl ketones to yield the corresponding cyanohydrins. The mutants (especially MeHNL-W128A) are in most cases superior to the wild-type (wt) enzyme when diisopropyl ether is used as the solvent. Substitution of tryptophan128 by an alanine residue enlarges the entrance channel to the active site of MeHNL and thus facilitates access of sterically demanding substrates to the active site, as clearly demonstrated for aromatic aldehydes, especially 3-phenoxybenzaldehyde. These experimental results are in accordance with the X-ray crystal structure of MeHNL-W128A. Aliphatic aldehydes, surprisingly, do not demonstrate this reactivity dependence of mutants on substrate bulkiness. Comparative reactions of 3-phenoxybenzaldehyde with wtMeHNL and MeHNL-W128A in both aqueous citrate buffer and a two-phase system of water/methyl tert-butyl ether again reveal the superiority of the mutant enzyme: 3-phenoxybenzaldehyde was converted quantitatively into a cyanohydrin nearly independently of the amount of enzyme present, with a space-time yield of 57 g L−1 h−1.