Enzyme Engineering Enables Inversion of Substrate Stereopreference of the Halogenase WelO5*

Abstract

Enzymatic late-stage diversification of small molecules has the potential to rapidly generate diversity in compound libraries dedicated to drug discovery. In this context, freestanding Fe(II)/ α-ketoglutarate-dependent halogenases have raised particular interest as this enzyme family allows the otherwise difficult regio-and stereoselective halogenation of unactivated C(sp 3 )À H bonds. Here, we report the development of two engineered variants of the halogenase WelO5* for the racemic resolution of a mixture of stereoisomers generated in the synthesis of a bioactive martinelline-derived fragment. By screening a 3-site combinatorial variant library, we could identify two variants exhibiting exquisite substrate selectivity towards the desired enantiomers. Strikingly, the inversion of substrate stereopreference between the halogenase variants was achieved by varying only three residues in the active site. Protein crystallization and subsequent structure elucidation of the wildtype enzyme and a WelO5* variant shed light on the factors governing substrate acceptance and selectivity.

A Collaborative Journey towards the Late‐Stage Functionalization of Added‐Value Chemicals Using Engineered Halogenases

“…We were able to gain better understanding of evolvability of Fe/αketoglutarate dependent enzymes and develop this promising enzyme class further. In only a few years, substantial results were obtained and resulted in several publications, [30,35,[65][66][67] highlighting the benefits of cross-industry and academic collaboration.…”

Discovery and substrate specificity engineering of nucleotide halogenases

“…αKGHs employ a high-valent iron-oxo intermediate to selectively activate sp 3 C−H bonds, generating a substrate radical that subsequently reacts with a halide ligand to yield the halogenated product 10,11 . A series of αKGHs acting on carrier-protein-tethered substrates (SyrB2 family) [12][13][14][15][16][17] , alkaloid natural products (WelO5 family and DAH) [18][19][20][21][22][23][24] , amino acids (BesD family) 25,26 , and nucleotides (AdaV family) 27,28 have been discovered and attracted extensive attention.…”