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

Research output: Contribution to journalArticlepeer-review

41 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, 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.

Original languageEnglish (US)
Pages (from-to)863-871
Number of pages9
JournalChemistry and Biology
Volume17
Issue number8
DOIs
StatePublished - 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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