Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates

Maika Genz, Okke Melse, Sandy Schmidt, Clare Vickers, Mark Dörr, Tom Van Den Bergh, Henk-Jan Joosten, Uwe T. Bornscheuer

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)3199-3202
Number of pages4
JournalChemCatChem
Volume8
Issue number20
DOIs
Publication statusPublished - 20 Oct 2016
Externally publishedYes

Fingerprint

Transaminases
Amines
amines
engineering
Substrates
Ketones
ketones
enzymes
Enzymes
Enantiomers
enantiomers
Bioinformatics
mutations
Drug products
Screening
screening
industries
Throughput
synthesis
Pharmaceutical Preparations

Keywords

  • amines
  • bioinformatics
  • enzyme catalysis
  • protein engineering
  • transaminases

ASJC Scopus subject areas

  • Catalysis
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Cite this

Genz, M., Melse, O., Schmidt, S., Vickers, C., Dörr, M., Van Den Bergh, T., ... Bornscheuer, U. T. (2016). Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates. ChemCatChem, 8(20), 3199-3202. https://doi.org/10.1002/cctc.201601007

Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates. / Genz, Maika; Melse, Okke; Schmidt, Sandy; Vickers, Clare; Dörr, Mark; Van Den Bergh, Tom; Joosten, Henk-Jan; Bornscheuer, Uwe T.

In: ChemCatChem, Vol. 8, No. 20, 20.10.2016, p. 3199-3202.

Research output: Contribution to journalArticleResearchpeer-review

Genz, M, Melse, O, Schmidt, S, Vickers, C, Dörr, M, Van Den Bergh, T, Joosten, H-J & Bornscheuer, UT 2016, 'Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates' ChemCatChem, vol. 8, no. 20, pp. 3199-3202. https://doi.org/10.1002/cctc.201601007
Genz, Maika ; Melse, Okke ; Schmidt, Sandy ; Vickers, Clare ; Dörr, Mark ; Van Den Bergh, Tom ; Joosten, Henk-Jan ; Bornscheuer, Uwe T. / Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates. In: ChemCatChem. 2016 ; Vol. 8, No. 20. pp. 3199-3202.
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