Here, we report the development of EnzyMS, a Python-based pipeline for the analysis of high-resolution liquid chromatography-mass spectrometry (LC-MS) data specifically tailored for biocatalysis experiments. Applying EnzyMS to biocatalytic reactions carried out with variants of Fe(II)/α-ketoglutarate-dependent halogenase WelO5∗ on the antifungal macrolide soraphen A, we discovered reaction outcomes that had not been observable when using standard analysis software. Interestingly, we detected a previously unreported selective oxidative demethylation of soraphen A alongside the reported hydroxylations and chlorinations. Building on this finding, a computationally guided protein engineering approach allowed us to identify a WelO5∗ variant that exhibited a 3-fold improved demethylation performance by only creating and testing three predicted variants. In summary, we showcase the utility of the EnzyMS workflow and its potential to enable rapid detection of previously unobserved biocatalytic products and highlight the valuable synergies between data science pipelines and the computational design of enzymes.

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(sp³)−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.