title: Increasing the reaction rate of hydroxynitrile lyase from Hevea brasiliensis toward mandelonitrile by copying active site residues from an esterase that accepts aromatic esters.
abstract: The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single-amino-acid-substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL-L121Y, with a kcat 4.2 times higher and high enantioselectivity. Site-saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, Biotechnology Institute, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 (USA); Institute of Chemistry, University of Rostock, Albert-Einstein-Strasse 3A, 18059 Rostock (Germany).
authors.forename: Jan
authors.initials: J
authors.lastname: von Langermann
authors.forename: David M
authors.initials: DM
authors.lastname: Nedrud
authors.forename: Romas J
authors.initials: RJ
authors.lastname: Kazlauskas
mesh_terms: D000097:Acetonitriles / Q000737:chemistry*; D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004950:Esterases / Q000737:chemistry*; D004952:Esters / Q000737:chemistry; D028482:Hevea / Q000201:enzymology*; D006841:Hydrocarbons, Aromatic / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry; D058767:Protein Unfolding; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000097:Acetonitriles; D004952:Esters; D006841:Hydrocarbons, Aromatic; D010940:Plant Proteins; C045516:mandelonitrile; C499506:SABP2 protein, Nicotiana tabacum; D004950:Esterases; D000446:Aldehyde-Lyases; C021541:mandelonitrile lyase
keywords: enzyme catalysis; esterases; hydroxynitrile lyase; promiscuous activity; protein engineering