期刊
CHEMCATCHEM
卷 13, 期 18, 页码 3914-3919出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cctc.202100591
关键词
alpha-ketoglutarate dependent oxygenases; biocatalysis; C-H functionalization; directed evolution; enzymatic halogenation
资金
- ZHAW
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.
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.
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