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Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor.
- Source :
-
Communications Chemistry . 2/28/2024, Vol. 7 Issue 1, p1-11. 11p. - Publication Year :
- 2024
-
Abstract
- 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 kcat 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. Ipatasertib is a potent Akt (protein kinase B) inhibitor synthesized via a chemoenzymatic process. Here, the authors use mutational scanning and algorithm-aided enzyme engineering to optimize a ketoreductase from Sporidiobolus salmonicolor and generate a 10-amino acid substituted variant exhibiting a 64-fold higher kcat and improved yield for the relevant alcohol intermediate, with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L−1 ketone after 30 h. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 23993669
- Volume :
- 7
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Communications Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 175829358
- Full Text :
- https://doi.org/10.1038/s42004-024-01130-5