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Increasing the Reaction Rate of Hydroxynitrile Lyase from Hevea brasiliensis toward Mandelonitrile by Copying Active Site Residues from an Esterase that Accepts Aromatic Esters
Author(s) -
von Langermann Jan,
Nedrud David M.,
Kazlauskas Romas J.
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402081
Subject(s) - hevea brasiliensis , active site , chemistry , stereochemistry , esterase , substrate (aquarium) , lyase , enzyme kinetics , saturated mutagenesis , organic chemistry , biochemistry , enzyme , biology , ecology , natural rubber , gene , mutant
The natural substrate of hydroxynitrile lyase from rubber tree ( Hb HNL, Hevea brasiliensis ) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of Hb HNL toward this unnatural substrate, we replaced active site residues in Hb 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 Hb HNL reacted more rapidly with mandelonitrile. The best was Hb HNL‐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.