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Computational redesign of an ω-transaminase from Pseudomonas jessenii for asymmetric synthesis of enantiopure bulky amines

DOI: 10.1021/acscatal.1c02053 DOI Help

Authors: Qinglong Meng (University of Groningen) , Carlos Ramírez-Palacios (University of Groningen) , Nikolas Capra (University of Groningen) , Mattijs E. Hooghwinkel (University of Groningen) , Sebastian Thallmair (University of Groningen) , Henriëtte J. Rozeboom (University of Groningen) , Andy-Mark W. H. Thunnissen (University of Groningen) , Hein J. Wijma (University of Groningen) , Siewert J. Marrink (University of Groningen) , Dick B. Janssen (University of Groningen)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: August 2021
Diamond Proposal Number(s): 19800

Open Access Open Access

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.

Journal Keywords: aminotransferase; substrate scope engineering; steric hindrance; green chemistry; computer-aided design; biocatalysis; protein

Diamond Keywords: Enzymes; Bacteria

Subject Areas: Biology and Bio-materials, Chemistry, Information and Communication Technology


Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Added On: 14/08/2021 21:38

Documents:
acscatal.1c02053.pdf

Discipline Tags:

Biochemistry Computing & software technologies Catalysis Information & Communication Technologies Chemistry Structural biology Chemical Engineering Engineering & Technology Organic Chemistry Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)