Abstract
AbstractNon‐heme iron/α‐ketoglutarate dependent halogenases acting on freestanding substrates catalyze 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 ofL‐prolinecis‐4‐hydroxylase fromSinorhizobium melilotiinto 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 ofL‐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 apparentkcat/Kmfor chlorination ofL‐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.
Highlights
The introduction of a halogen into a molecule’s scaffold can alter its bioactivity or act as a useful synthetic handle for diversification and modification of late-stage synthetic [b] Dr T
The recently discovered non heme iron/αketoglutarate (Fe/αKG) halogenases, e. g., WelO5,[3] WelO5*,[4] Wi-WelO15,[5] AmbO5[6] and BesD,[7] acting on free-standing substrates have broadened the biocatalytic potential of radical halogenation by overcoming the complexity barrier of previously known Fe/αKG halogenases requiring carrier-proteintethered substrates (BarB1 and BarB2,[8] SyrB2,[9] CytC3,[10] CmaB,[11] HctB,[12] CurA,[13] KthP[14])
Fe/αKG halogenases belong to the superfamily of α-ketoglutarate-dependent oxygenases which coordinate the co-factor Fe(II) by two conserved histidine residues, a carboxylate residue and the cofactor α-ketoglutarate in their active site
Summary
The introduction of a halogen into a molecule’s scaffold can alter its bioactivity or act as a useful synthetic handle for diversification and modification of late-stage synthetic [b] Dr T.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
