Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor.

Autor: Honda Malca S; Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland., Duss N; Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland., Meierhofer J; Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland.; Analytical Research and Development, MSD Werthenstein BioPharma GmbH, Industrie Nord 1, 6105 Schachen, Switzerland., Patsch D; Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland., Niklaus M; Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland., Reiter S; Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland.; Manufacturing Science and Technology, Fisher Clinical Services GmbH, Biotech Innovation Park, 2543 Lengnau, Switzerland., Hanlon SP; Process Chemistry and Catalysis, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland., Wetzl D; Process Chemistry and Catalysis, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland.; Nonclinical Drug Development, Boehringer Ingelheim International GmbH, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany., Kuhn B; Pharmaceutical Research and Early Development, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland., Iding H; Process Chemistry and Catalysis, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland., Buller R; Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland. rebecca.buller@zhaw.ch.
Jazyk: angličtina
Zdroj: Communications chemistry [Commun Chem] 2024 Feb 28; Vol. 7 (1), pp. 46. Date of Electronic Publication: 2024 Feb 28.
DOI: 10.1038/s42004-024-01130-5
Abstrakt: Semi-rational enzyme engineering is a powerful method to develop industrial biocatalysts. Profiting from advances in molecular biology and bioinformatics, semi-rational approaches can effectively accelerate enzyme engineering campaigns. Here, we present the optimization of a ketoreductase from Sporidiobolus salmonicolor for the chemo-enzymatic synthesis of ipatasertib, a potent protein kinase B inhibitor. Harnessing the power of mutational scanning and structure-guided rational design, we created a 10-amino acid substituted variant exhibiting a 64-fold higher apparent k cat and improved robustness under process conditions compared to the wild-type enzyme. In addition, the benefit of algorithm-aided enzyme engineering was studied to derive correlations in protein sequence-function data, and it was found that the applied Gaussian processes allowed us to reduce enzyme library size. The final scalable and high performing biocatalytic process yielded the alcohol intermediate with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L -1 ketone after 30 h. Modelling and kinetic studies shed light on the mechanistic factors governing the improved reaction outcome, with mutations T134V, A238K, M242W and Q245S exerting the most beneficial effect on reduction activity towards the target ketone.
(© 2024. The Author(s).)
Databáze: MEDLINE
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