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Engineering the Amine Transaminase from Vibrio fluvialis towards Branched‐Chain Substrates
Author(s) -
Genz Maika,
Melse Okke,
Schmidt Sandy,
Vickers Clare,
Dörr Mark,
van den Bergh Tom,
Joosten HenkJan,
Bornscheuer Uwe T.
Publication year - 2016
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201601007
Subject(s) - amine gas treating , enantiomeric excess , chemistry , in silico , combinatorial chemistry , yield (engineering) , ketone , enantiomer , enzyme , stereochemistry , directed evolution , transaminase , substrate (aquarium) , enantioselective synthesis , biocatalysis , mutant , organic chemistry , biochemistry , biology , catalysis , materials science , gene , reaction mechanism , ecology , metallurgy
Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATAs) are versatile enzymes to synthesize chiral amines, the wildtype enzymes do not accept ketones with two large substituents next to the carbonyl functionality. Using bioinformatic tools to design a seven‐site mutant library followed by high‐throughput screening, we were able to identify variants of the enzyme from Vibrio fluvialis (VF‐ATA) with a widened binding pocket, as exemplified for a range of ketones. Three variants allowed the asymmetric synthesis of 2,2‐dimethyl‐1‐phenylpropan‐1‐amine—not accessible by any wildtype ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100 % conversion of the ketone to yield the amine with an enantiomeric excess value >99 %, notably with preference for the ( R )‐enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved pocket and, hence, allowed explanation of the experimental results.