Increasing the reaction rate of hydroxynitrile lyase from hevea brasiliensis toward mandelonitrile by copying active site residues from an esterase that accepts aromatic esters

Jan Von Langermann, David M. Nedrud, Romas J. Kazlauskas

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single-amino-acid-substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL-L121Y, with a k cat 4.2 times higher and high enantioselectivity. Site-saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively. A tight squeeze: The unnatural substrate mandelonitrile reacts approximately four times more rapidly with variants of hydroxynitrile lyase that contain smaller active sites. Modeling indicated that the substitutions in these variants reduce the size of the substrate-binding site and favor a productive orientation of the substrate.

Original languageEnglish (US)
Pages (from-to)1931-1938
Number of pages8
JournalChemBioChem
Volume15
Issue number13
DOIs
StatePublished - Jan 1 2014

Keywords

  • enzyme catalysis
  • esterases
  • hydroxynitrile lyase
  • promiscuous activity
  • protein engineering

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