TY - JOUR
T1 - Computational Redesign of an ω-Transaminase from Pseudomonas jessenii for Asymmetric Synthesis of Enantiopure Bulky Amines
AU - Meng, Qinglong
AU - Ramírez-Palacios, Carlos
AU - Capra, Nikolas
AU - Hooghwinkel, Mattijs E.
AU - Thallmair, Sebastian
AU - Rozeboom, Henriëtte J.
AU - Thunnissen, Andy Mark W.H.
AU - Wijma, Hein J.
AU - Marrink, Siewert J.
AU - Janssen, Dick B.
N1 - Funding Information:
Q.M. thanks the China Scholarship Council for a Ph.D. fellowship. C.R.P. thanks CONACYT for the doctoral fellowship. Part of this project has received funding from the European Union’s Horizon 2020 Programme (Marie Curie Actions-ITN ES-Cat) under GA no. 722610, which supported N.C. The research of H.J.W. was supported by the Dutch Ministry of Economic Affairs through BE-Basic, grant FS02.005. We thank the Center for Information Technology of the University of Groningen for providing access to the Peregrine high-performance computing cluster. We thank the staff of Diamond Light Source at Oxford for the excellent support and beamtime allocation.
Publisher Copyright:
© 2021 The Authors. Published by American Chemical Society.
PY - 2021/8
Y1 - 2021/8
N2 - 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.
AB - 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.
KW - aminotransferase
KW - biocatalysis
KW - computer-aided design
KW - green chemistry
KW - protein engineering
KW - steric hindrance
KW - substrate scope engineering
UR - http://www.scopus.com/inward/record.url?scp=85113904504&partnerID=8YFLogxK
U2 - 10.1021/acscatal.1c02053
DO - 10.1021/acscatal.1c02053
M3 - Article
AN - SCOPUS:85113904504
SN - 2155-5435
VL - 11
SP - 10733
EP - 10747
JO - ACS Catalysis
JF - ACS Catalysis
IS - 17
ER -