Non‐heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp3‐hybridized carbon centers with high stereo‐ and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline‐derived fragment. Using structure‐guided evolution, a halogenase variant with a more than 290‐fold higher total turnover number and a 400‐fold higher apparent kcat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non‐heme iron halogenase beyond the native indole‐alkaloid‐type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late‐stage halogenation.

Biocatalysis is an effective tool to access chiral molecules that are otherwise hard to synthesize or purify. Time-efficient processes are needed to develop enzymes that adequately perform the desired chemistry. We evaluated machine-directed evolution as an enzyme engineering strategy using a moderately stereoselective imine reductase as the model system. We compared machine-directed evolution approaches to deep mutational scanning (DMS) and error-prone PCR. Within one cycle, it was found that machine-directed evolution yielded a library of high-activity mutants with a dramatically shifted activity distribution compared to that of traditional directed evolution. Structure-guided analysis revealed that linear additivity might provide a simple explanation for the effectiveness of machine-directed evolution. The most active and selective enzyme mutant, which was identified through DMS and error-prone PCR, was used for the gram-scale synthesis of the H4 receptor antagonist ZPL389 with full conversion, > 99% ee (R), and a 72% yield.

Modification of aliphatic C−H bonds in a regio-and stereoselective manner can pose a formidable challenge in organic chemistry. In this context, the use of nonheme iron and α-ketoglutarate-dependent dioxygenases (αKGDs) represents an interesting tool for C−H activation as this enzyme family can catalyze a broad set of synthetically valuable reactions including hydroxylations, oxidations, and desaturations. The consensus reaction mechanism of αKGDs proceeds via the formation of a Fe(IV)-oxo complex capable of hydrogen atom transfer (HAT) from an sp 3 -hybridized substrate carbon center. The resulting substrate radical and Fe(III)−OH cofactor are considered to be the branch point toward the possible reaction outcomes which are determined by the enzyme's active site architecture. To date, the modulation of the reaction fate in Fe(II)/α-ketoglutarate-dependent dioxygenases via enzyme engineering has been mainly elusive. In this study, we therefore set out to engineer the L-proline cis-4-hydroxylase SmP4H from Sinorhizobium meliloti for selective oxidative modifications of the nonproteinogenic amino acid L-homophenylalanine (L-hPhe) to produce pharmacological relevant small molecule intermediates. Using structure-guided directed evolution, we improved the total turnover number, the k cat , as well as the k cat /K m of the hydroxylation reaction yielding the desired γ-hydroxylation product by approximately 10-fold, >100-fold, and >300-fold, respectively. Notably, the exchange of only one amino acid in the active site (W40Y) allowed us to reprogram the natural hydroxylase to predominantly act as a desaturase, presumably through tyrosine's capability to serve as a catalytic entity in the reaction mechanism. An investigation of the substrate scope revealed additional acceptance of the noncanonical amino acids Lhomotyrosine and (S)-α-amino-3,4-dichlorobenzenebutanoic acid by SmP4H variants.

Cladosporin, a natural product known for decades, has recently been discovered to display potent and selective antiplasmodial activity by inhibition of lysyl‐tRNA synthetase. It was subjected to a panel of oxidative biotransformations with one fungal and two actinomycetes strains, as well as a triple mutant bacterial CYP102A1, yielding eight, mostly hydroxylated, derivatives. These new compounds covered a wide chemical space and contained two pairs of epimers in the tetrahydropyran ring. Although less potent than the parent compound, all analogues showed activity in a cell‐based synthetase assay, thus demonstrating uptake and on‐target activity in living cells with varying degrees of selectivity for the enzyme lysyl‐tRNA synthetase from Plasmodium falciparum and highlighting sites suitable for synthesis of future cladosporin analogues. Compounds with adjacent hydroxy functions showed different MS/MS fragmentation that can be explained in terms of an, in some cases, regioselective loss of water followed by a retro‐Diels–Alder reaction.

Nicht-Häm-Eisen(II)-Halogenasen sind wertvolle Biokatalysatoren fürd ie stereo-und enantioselektive Halogenierung von nicht-aktivierten sp 3 -Kohlenstoffatomen. Das Substratspektrum dieser Enzyme ist jedocheng auf natürliche Substrate beschränkt. Hier haben wir die Halogenase WelO5* zur Chlorierung eines pharmazeutisch interessanten Martinellin-Fragments verändert. Durch Evolution konnten wir eine Halogenase entwickeln, die eine mehr als 290-fachh çhere katalytische Produktivitätu nd 400-fachh çhere scheinbare Wechselzahl (app.k cat )a ls der Wildtyp aufweist. Wirk onnten Schlüsselpositionen im aktiven Zentrum identifizieren, die die Derivatisierung unterschiedlicher Positionen im Substrat erlauben. Wirl iefern das erste Beispiel fürdas Engineering von Nicht-Häm-Eisen(II)-Halogenasen zur Erweiterung des Substratspektrums über die nativen Indolalkaloide hinaus.D ie Evolvierbarkeit von WelO5* unterstreicht den Nutzen dieser Enzymfamilie fürd ie Halogenierung von komplexen Chemikalien.