Increasing the Reaction Rate of Hydroxynitrile Lyase from Hevea brasiliensis toward Mandelonitrile by Copying Active Site Residues from an Esterase that Accepts Aromatic Esters. Issue 13 (18th July 2014)

• Record Type: Journal Article
• Authors: von Langermann , Jan; Nedrud, David M.; Kazlauskas, Romas J.
• Is Part Of: Chembiochem. Volume 15:Issue 13(2014)
• Page Start: 1931
• Page End: 1938
• Publication Date: 2014-07-18
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201402081
• Languages: English
• ISSNs: 1439-4227

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p>The natural substrate of hydroxynitrile lyase from rubber tree (<italic>Hb</italic>HNL, <italic>Hevea brasiliensis</italic>) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of <italic>Hb</italic>HNL toward this unnatural substrate, we replaced active site residues in <italic>Hb</italic>HNL 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 <italic>Hb</italic>HNL reacted more rapidly with mandelonitrile. The best was <italic>Hb</italic>HNL‐L121Y, with a <italic>k</italic>_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.</p> </abstract>