Protein Science 2001-05-01

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

H Lauble, B Miehlich, S Förster, H Wajant, F Effenberger

Index: Protein Sci. 10(5) , 1015-22, (2001)

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Abstract

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


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