Switching from an esterase to a hydroxynitrile lyase mechanism requires only two amino acid substitutions.
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.
Padhi
Santosh Kumar
SK
Department of Biochemistry, Molecular Biology, and Biophysics, and the Biotechnology Institute, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108, USA.
Fujii
Ryota
R
Legatt
Graig A
GA
Fossum
Sara L
SL
Berchtold
Reto
R
Kazlauskas
Romas J
RJ
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
D004950:Esterases; D000446:Aldehyde-Lyases