title: Switching from an esterase to a hydroxynitrile lyase mechanism requires only two amino acid substitutions.
abstract: The alpha/beta hydrolase superfamily contains mainly esterases, which catalyze hydrolysis, but also includes hydroxynitrile lyases, which catalyze addition of cyanide to aldehydes, a carbon-carbon bond formation. Here, we convert a plant esterase, SABP2, into a hydroxynitrile lyase using just two amino acid substitutions. Variant SABP2-G12T-M239K lost the ability to catalyze ester hydrolysis (<0.9 mU/mg) and gained the ability to catalyze the release of cyanide from mandelonitrile (20 mU/mg, k(cat)/K(M) = 70 min(-1)M(-1)). This variant also catalyzed the reverse reaction, formation of mandelonitrile with low enantioselectivity: 20% ee (S), E = 1.5. The specificity constant for the lysis of mandelontrile is 13,000-fold faster than the uncatalyzed reaction and only 1300-fold less efficient (k(cat/)K(M)) than hydroxynitrile lyase from rubber tree.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, and the Biotechnology Institute, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108, USA.
authors.forename: Santosh Kumar
authors.initials: SK
authors.lastname: Padhi
authors.forename: Ryota
authors.initials: R
authors.lastname: Fujii
authors.forename: Graig A
authors.initials: GA
authors.lastname: Legatt
authors.forename: Sara L
authors.initials: SL
authors.lastname: Fossum
authors.forename: Reto
authors.initials: R
authors.lastname: Berchtold
authors.forename: Romas J
authors.initials: RJ
authors.lastname: Kazlauskas
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D028482:Hevea / Q000201:enzymology; D008958:Models, Molecular; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D004950:Esterases; D000446:Aldehyde-Lyases