Computational Redesign of an ω-Transaminase from Pseudomonas jessenii for Asymmetric Synthesis of Enantiopure Bulky Amines

Qinglong Meng, Carlos Ramírez-Palacios, Nikolas Capra, Mattijs E. Hooghwinkel, Sebastian Thallmair, Henriëtte J. Rozeboom, Andy Mark W.H. Thunnissen, Hein J. Wijma, Siewert J. Marrink, Dick B. Janssen*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

27 Citations (Scopus)
126 Downloads (Pure)

Abstract

ω-Transaminases (ω-TA) are attractive biocatalysts for the production of chiral amines from prochiral ketones via asymmetric synthesis. However, the substrate scope of ω-TAs is usually limited due to steric hindrance at the active site pockets. We explored a protein engineering strategy using computational design to expand the substrate scope of an (S)-selective ω-TA from Pseudomonas jessenii (PjTA-R6) toward the production of bulky amines. PjTA-R6 is attractive for use in applied biocatalysis due to its thermostability, tolerance to organic solvents, and acceptance of high concentrations of isopropylamine as amino donor. PjTA-R6 showed no detectable activity for the synthesis of six bicyclic or bulky amines targeted in this study. Six small libraries composed of 7-18 variants each were separately designed via computational methods and tested in the laboratory for ketone to amine conversion. In each library, the vast majority of the variants displayed the desired activity, and of the 40 different designs, 38 produced the target amine in good yield with >99% enantiomeric excess. This shows that the substrate scope and enantioselectivity of PjTA mutants could be predicted in silico with high accuracy. The single mutant W58G showed the best performance in the synthesis of five structurally similar bulky amines containing the indan and tetralin moieties. The best variant for the other bulky amine, 1-phenylbutylamine, was the triple mutant W58M + F86L + R417L, indicating that Trp58 is a key residue in the large binding pocket for PjTA-R6 redesign. Crystal structures of the two best variants confirmed the computationally predicted structures. The results show that computational design can be an efficient approach to rapidly expand the substrate scope of ω-TAs to produce enantiopure bulky amines.

Original languageEnglish
Pages (from-to)10733-10747
Number of pages15
JournalACS Catalysis
Volume11
Issue number17
DOIs
Publication statusPublished - Aug-2021

Keywords

  • aminotransferase
  • biocatalysis
  • computer-aided design
  • green chemistry
  • protein engineering
  • steric hindrance
  • substrate scope engineering

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