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.