Structure and Molecular Mechanism of Hydroxnitrile Lyases

Hydroxynitrile lyases (HNLs) catalyze the cyanohydrin reaction, i.e. the reversible cleavage of a-cyanohydrins to yield hydrocyanic acid and the corresponding aldehyde or ketone. Biologically, they form part of a vegetal defense system, exploiting the toxicity of hydrocyanic acid. HNLs show a large natural diversity, i.e. several different HNLs have developed by convergent evolution from ancestors belonging to entirely different protein superfamilies. The very different active site architectures in these unrelated enzymes allows to study the structural requirements for catalysis of cyanohydrin cleavage. HNLs are currently being used as industrial biocatalysts for the enantioselective synthesis of chiral cyanohydrins at the level of hundreds of metric tons per year. Enzymes with (R) and (S) enantioselectivity are available as a result of the large natural diversity of HNLs. 3D structural data are available (largely as a result of research from the applicants laboratory) for 3 types of HNL, related a/ß hydrolases, FAD-dependent oxidoreductases and carboxypeptidases, respectively. The details of the molecular mechanism of enzyme catalysis, however, has so far only been elucidated for HNLs belonging to the class of a/ß hydrolases. The first part of the present application deals with structural and functional experiments to elucidate the molecular mechanism of the HNL from almond (Prunus amygdalus), whose native 3D structure has recently been elucidated in the applicants laboratory. We propose the crystallographic structure elucidation of mutants, of enzyme variants with a reduced cofactor and of enzyme-substrate complexes. The structural studies will be complemented by functional studies. Besides its inherent scientific interest, the mechanistic insight will be the basis for designing enzymes with improved properties for industrial biocatalysis. Secondly, the crystallographic elucidation of the 3D structure of the HNL from flax (Linum usitatissimum) is proposed. This HNL is related to zinc-dependent alcohol dehydrogenases, and forms a novel and as yet structurally uncharacterized type of HNL. Elucidation of its native 3D structure will be followed by structural and functional studies on mutants and complexes to deduce the molecular mechanism. Finally, we propose to clone, overexpress and purify two completely novel types of HNL from the ferns Phlebodium aureum and Pteridium aquilinum, which will then be subjected to crystallographic analysis of their 3D structure, followed by mechanistic studies as above.

Hydroxynitrile lyases (HNLs) catalyze the cyanohydrin reaction, i.e. the reversible cleavage of a-cyanohydrins to yield hydrocyanic acid and the corresponding aldehyde or ketone. Biologically, they form part of a vegetal defense system, exploiting the toxicity of hydrocyanic acid. HNL`s show a large natural diversity, i.e. several different HNL`s have developed by convergent evolution from ancestors belonging to entirely different protein superfamilies. The very different active site architectures in these unrelated enzymes allows to study the structural requirements for catalysis of cyanohydrin cleavage. HNLs are currently being used as industrial biocatalysts for the enantioselective synthesis of chiral cyanohydrins at the level of hundreds of metric tons per year. Enzymes with (R) and (S) enantioselectivity are available as a result of the large natural diversity of HNLs. 3D structural data are available (largely as a result of research from the applicant`s laboratory) for 3 types of HNL, related a/ß hydrolases, FAD-dependent oxidoreductases and carboxypeptidases, respectively. The details of the molecular mechanism of enzyme catalysis, however, has so far only been elucidated for HNL`s belonging to the class of a/ß hydrolases. The first part of the present application deals with structural and functional experiments to elucidate the molecular mechanism of the HNL from almond (Prunus amygdalus), whose native 3D structure has recently been elucidated in the applicant`s laboratory. We propose the crystallographic structure elucidation of mutants, of enzyme variants with a reduced cofactor and of enzyme-substrate complexes. The structural studies will be complemented by functional studies. Besides its inherent scientific interest, the mechanistic insight will be the basis for designing enzymes with improved properties for industrial biocatalysis. Secondly, the crystallographic elucidation of the 3D structure of the HNL from flax (Linum usitatissimum) is proposed. This HNL is related to zinc-dependent alcohol dehydrogenases, and forms a novel and as yet structurally uncharacterized type of HNL. Elucidation of its native 3D structure will be followed by structural and functional studies on mutants and complexes to deduce the molecular mechanism. Finally, we propose to clone, overexpress and purify two completely novel types of HNL from the ferns Phlebodium aureum and Pteridium aquilinum, which will then be subjected to crystallographic analysis of their 3D structure, followed by mechanistic studies as above.