Re-Programming and Optimization of a L-Proline cis-4-Hydroxylase for the cis-3-Halogenation of its Native Substrate

Non-heme iron/alpha-ketoglutarate dependent halogenases acting on freestanding substrates catalyze the regio- and stereoselective halogenation of inactivated C(sp(3))-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 (D108G) into the enzyme's active site. 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 C4-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 k(cat)/K-m 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/alpha KG dependent dioxygenases for C-H functionalization.

Automated summary

By introducing a single point mutation, L-proline cis-4-hydroxylase was successfully redesigned into a halogenase with optimized regio-divergent reaction chemistry. Through several rounds of directed evolution, an optimized halogenase variant with 98-fold improved apparent k(cat)/K-m for chlorination was identified, showcasing the potential of non-heme Fe/alpha KG dependent dioxygenases for C-H functionalization.