Papadopoulou, Athena, Meierhofer, Jasmin, Meyer, Fabian, Hayashi, Takahiro, Schneider, Samuel, Sager, Emine and Rebecca, Buller (2021) Re-programming and optimization of a L-proline cis-4-hydroxylase for the cis-3-halogenation of its native substrate. ChemCatChem.. ISSN 1867-38801867-3899

Abstract

Freestanding non-heme iron/α-ketoglutarate dependent halogenases enable the regio- and stereoselective halogenation of inactivated C(sp3)-H bonds. Yet, with only a handful of these halogenases characterized, the biosynthetic potential of enzymatic radical halogenation remains limited. Herein, we describe the remodeling of L-proline cis-4-hydroxylase from Sinorhizobium meliloti into a halogenase by introduction of a single point mutation into the enzyme’s active site (D108G). The re-programmed halogenase displays a striking regio-divergent reaction chemistry: While halogenation of L-proline exclusively occurs at the C3-position, the retained hydroxylation activity leads to derivatization at the C-4 position, corresponding to the regioselectivity of the wildtype enzyme. By employing several rounds of directed evolution, an optimized halogenase variant with 98-fold improved apparent kcat / Km for chlorination of L-proline compared to the parental enzyme SmP4H (D108G) was identified. The development and optimization of this novel halogenation biocatalyst highlights the possibility to rationally harness the chemical versatility of non-heme Fe/αKG dependent dioxygenases for C-H functionalization.

Item Type:	Article
Keywords:	α-ketoglutarate dependent oxygenases • directed evolution • halogenation • biocatalysis • C−H functionalization
Date Deposited:	07 Sep 2021 00:45
Last Modified:	07 Sep 2021 00:45
URI:	https://oak.novartis.com/id/eprint/44751