Switching from an esterase to a hydroxynitrile lyase mechanism requires only two amino acid substitutions

Santosh Kumar Padhi; Ryota Fujii; Graig A. Legatt; Sara L. Fossum; Reto Berchtold; Romas J. Kazlauskas

doi:10.1016/j.chembiol.2010.06.013

Switching from an esterase to a hydroxynitrile lyase mechanism requires only two amino acid substitutions

Santosh Kumar Padhi, Ryota Fujii, Graig A. Legatt, Sara L. Fossum, Reto Berchtold, Romas J. Kazlauskas

Biotechnology Institute

Research output: Contribution to journal › Article › peer-review

44 Scopus citations

Abstract

The α/β 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^-1M^-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.

Original language	English (US)
Pages (from-to)	863-871
Number of pages	9
Journal	Chemistry and Biology
Volume	17
Issue number	8
DOIs	https://doi.org/10.1016/j.chembiol.2010.06.013
State	Published - Aug 27 2010

Bibliographical note

Funding Information:
We thank Seongsoon Park, David Soriano Del Amo, and Tobias Ölander for initial experiments, Uwe T. Bornscheuer (U. Greifswald, Germany) for helpful suggestions, Daniel Klessig (Boyce Thompson Institute for Plant Research, Ithaca, NY) for the plasmid encoding SABP2, the U.S. National Science Foundation; Grant Number: CHE-0616560 for financial support and the Minnesota Supercomputing Institute for access to software and computers and a summer internship to S.L.F.

Keywords

CHEMBIO
PROTEINS

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abstract = "The α/β 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, kcat/KM = 70 min-1M-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/KM) than hydroxynitrile lyase from rubber tree.",

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note = "Funding Information: We thank Seongsoon Park, David Soriano Del Amo, and Tobias {\"O}lander for initial experiments, Uwe T. Bornscheuer (U. Greifswald, Germany) for helpful suggestions, Daniel Klessig (Boyce Thompson Institute for Plant Research, Ithaca, NY) for the plasmid encoding SABP2, the U.S. National Science Foundation; Grant Number: CHE-0616560 for financial support and the Minnesota Supercomputing Institute for access to software and computers and a summer internship to S.L.F. ",

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AU - Berchtold, Reto

AU - Kazlauskas, Romas J.

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N2 - The α/β 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, kcat/KM = 70 min-1M-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/KM) than hydroxynitrile lyase from rubber tree.

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Switching from an esterase to a hydroxynitrile lyase mechanism requires only two amino acid substitutions

Abstract

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