Catalytic anti-Markovnikov oxidation of alkene feedstocks could simplify synthetic routes to many important molecules and solve a long-standing challenge in chemistry. Here we report the engineering of a cytochrome P450 enzyme by directed evolution to catalyze metal-oxo-mediated anti-Markovnikov oxidation of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidation over the kinetically favored alkene epoxidation by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions.

Biocatalytic halogenation with tryptophan halogenases is hampered by severe limitations such as low activity and stability. These drawbacks can be overcome by directed evolution, but for screening large mutant libraries, a facile high-throughput method is required. Therefore, we developed a quantitative halogenase assay based on a Suzuki-Miyaura cross-coupling towards the formation of a fluorescent aryltryptophan. The technique was optimized for application in crude E. coli lysate without intermediary purification steps, and was used for quantitatively monitoring the formation of halogenated tryptophans with high specificity by facile fluorescence screening in microtiter plates. This novel screening approach was exploited to engineer a thermostable tryptophan 6-halogenase. Libraries were constructed by error-prone PCR and selected for improved thermal resistance simply by fluorogenic cross-coupling. Our method led to an enzyme variant with substantially increased thermal stability and 2.5-fold improved activity.

Edited by Ruma BanerjeeFlavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH 2 , oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate tryptophan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 Å resolution. The structure revealed that the C6 of tryptophan is positioned next to the ⑀-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of L-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases.The authors declare that they have no conflicts of interest with the contents of this article. This article contains Tables S1 and S2 and Figs. S1-S17. The atomic coordinates and structure factors (codes 6H43, 6H44, and 6IB5) have been deposited in the Protein Data Bank (http://wwpdb.org/). . 3 The abbreviations used are: FDH, flavin-dependent halogenase; aa, amino acid(s); ESI, electrospray ionization; HSQC, heteronuclear single-quantum coherence; r.m.s.d., root mean square deviation; ROESY, rotating frame nuclear Overhauser effect spectroscopy; Bicine, N,N-bis(2-hydroxyethyl)-glycine; H-bond, hydrogen bond; Ni-NTA, nickel-nitrilotriacetic acid; RR-ADH, Rhodococcus ruber alcohol dehydrogenase; PDB, Protein Data Bank.Figure 8. Steric conflicts upon placing L-Trp from Trp-RebH in Trp-Thal and vice versa. Minor clashes are indicated as small green hexagons, and more severe clashes are shown as larger red hexagons. a, L-Trp from Trp-RebH (PDB code 2E4G) was placed in Trp-Thal. Clashes mainly occur between the L-Trp indole and the side chains of conserved residues Phe-112 and Trp-466. b, L-Trp from Trp-Thal was placed in Trp-RebH (PDB code 2E4G). Clashes mainly occur between the L-Trp indole and the side chain of Asn-470 and between the L-Trp carboxylate and the side chain...

The combination of the biocatalytic halogenation of l‐tryptophan with subsequent chemocatalytic Suzuki–Miyaura cross‐coupling reactions leads to the modular synthesis of an array of C5, C6, or C7 aryl‐substituted tryptophan derivatives. In a three‐step one‐pot reaction, the bromo substituent is initially incorporated regioselectively by immobilized tryptophan 5‐, 6‐, or 7‐halogenases, respectively, with concomitant cofactor regeneration. The halogenation proceeds in aqueous media at room temperature in the presence of NaBr and O2. After the separation of the biocatalyst by filtration, a Pd catalyst, base, and boronic acid are added to the aryl halide formed in situ to effect direct Suzuki–Miyaura cross‐coupling reactions followed by tert‐butoxycarbonyl (Boc) protection. After a single purification step, different Boc‐protected aryl tryptophan derivatives are obtained that can, for example, be used for peptide or peptidomimetic synthesis.

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