Mechanistic aspects of cyanogenesis from active-site mutant Ser80Ala of hydroxynitrile lyase from Manihot esculenta in complex with acetone cyanohydrin

The structure and function of hydroxynitrile lyase from Manihot esculenta ( MeHNL) have been analyzed by X-ray crystallography and site-directed mutage nesis. The crystal structure of the MeHNL-S80A mutant enzyme has been refin ed to an R-factor of 18.0% against diffraction data to 2.1-Angstrom resolut ion. The three-dimensional structure of the MeHNL-S80A-acetone cyanohydrin complex was determined at 2.2-Angstrom resolution and refined to an R-facto r of 18.7%. Thr11 and Cys81 involved in substrate binding have been substit uted by Ala in site-directed mutagenesis. The kinetic measurements of these mutant enzymes are presented. Combined with structural data, the results s upport a mechanism for cyanogenesis in which His236 as a general base abstr acts a proton from Ser80, thereby allowing proton transfer from the hydroxy l group of acetone cyanohydrin to Ser80. The His236 imidazolium cation then facilitates the leaving of the nitrile group by proton donating.