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Engineering of Saccharomyces pastorianus Old Yellow Enzyme 1 for the Synthesis of Pharmacologically Active (S)-Profen Derivatives

preprint
submitted on 29.08.2020 and posted on 31.08.2020 by Guigao Liu, Shang Li, Qinghua Shi, Hengyu Li, Jiyang Guo, Jingping Ouyang, Xian Jia, Lihan Zhang, Song You, Bin Qin
2-Arylpropionic acid derivatives, such as ibuprofen, constitute an important group of non-steroidal anti-inflammatory drugs (NSAIDs). Biocatalytic asymmetric reduction of 2-arylacrylic acid derivatives by ene reductases (EREDs) is a valuable approach for synthesis of these derivatives. However, previous bioreduction of 2-arylacrylic acid derivatives by either ERED wild-types or variants resulted solely in nonpharmacological (R)-enantiomers as the products. Here, we present the engineering of Saccharomyces pastorianus old yellow enzyme 1 (OYE1) into (S)-stereoselective enzymes, which afford pharmacologically active (S)-profen derivatives. By structural comparison of substrate recognition in related EREDs and analysis of non-covalent contacts in the pro-S model of OYE1, the key residues of OYE1 that switch its stereoselectivity to an (S)-stereopreference were identified. Systematic site-directed mutagenesis screening at these positions successfully provided the (S)-stereoselective OYE1 variants, which catalyzed stereoselective bioreduction of various profen precursors to afford pharmacologically active (S)-derivatives including (S)-ibuprofen and (S)-naproxen methyl esters with up to >99% ee values. Moreover, the key residues and mutation strategy obtained from OYE1 could be further transferred to OYE 2.6 (from Pichia stipitis) and KnOYE1 (from Kazachstania naganishii) to create the (S)-stereoselective EREDs. Our results may provide a generalizable strategy for stereocontrol of OYEs and set the basis for biocatalytic production of (S)-profens.

History

Email Address of Submitting Author

to-qinbin@163.com

Institution

Shenyang Pharmaceutical University

Country

China

ORCID For Submitting Author

0000-0002-9180-0550

Declaration of Conflict of Interest

No Conflicts of Interest

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