Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta |
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| Authors: | Hanspeter Lauble, Burkhard Miehlich, Siegfried F rster, Christoph Kobler, Harald Wajant, Franz Effenberger |
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| Affiliation: | 1.Institut für Organische Chemie der Universität Stuttgart, D-70569 Stuttgart, Germany ;2.Institut für Zellbiologie und Immunologie der Universität Stuttgart, D-70569 Stuttgart, Germany |
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| 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. |
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| Keywords: | Substrate specificity active-site tunnel mutant crystal structure |
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