title: Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convertHevea brasiliensishydroxynitrile lyase into an esterase.
abstract: Hevea brasiliensishydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues inHbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions inHbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat~ 0.1 min-1for hydrolysis ofp-nitrophenyl acetate. Adding a third substitution - Glu79His - increased esterase activity more than tenfold to kcat~ 1.6 min-1. The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity inHbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at the corresponding location ofCandidalipases is uncharged due to other active site differences and does not cause the same distortion. This example of the fine tuning of the same catalytic triad for different types of catalysis by subtle interactions with other active site residues shows how difficult it is to design new catalytic reactions of enzymes.
authors.affiliation: University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Nedrud
authors.affiliation: University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Lin
authors.affiliation: University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
authors.forename: Gilsinia
authors.initials: G
authors.lastname: Lopez
authors.affiliation: University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
authors.forename: Santosh K
authors.initials: SK
authors.lastname: Padhi
authors.affiliation: University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
authors.forename: Graig A
authors.initials: GA
authors.lastname: Legatt
authors.affiliation: University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
authors.forename: Romas J
authors.initials: RJ
authors.lastname: Kaz-Lauskas
keywords: catalytic promiscuity; divergent evolution; esterase; hydroxynitrile lyase; protein engineering; α/β-hydrolase