Evolvierte aliphatische Halogenasen ermöglichen die regiokomplementäre C‐H‐Funktionalisierung einer hochwertigen Chemikalie

Abstract

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.

Exploring the Biocatalytic Potential of Fe/α‐Ketoglutarate‐Dependent Halogenases

“…Turning to nature, the use of Fe/a-ketoglutarate-dependenth alogenases may offer av iable strategy toward the asymmetrich alogenation of unactivated CÀH bonds.F irst members of this family acting on freestanding substrates have been discovered and pioneering protein engineering studies suggest that the substrate scope of freestanding aKGHs can be more easily broadened than anticipated. [34,37] Nevertheless, the young field hast ot ackle ar ange of remaining challenges to bring Fe/a-ketoglutarate-dependent halogenases to their full potential. Most important will be to increase the low turnover number of the short-lived aKGHs, which likely suffer from autoxidation.…”

“…To wards this goal, structural studies of substrate-bound WelO5* and Wi-WelO15 variantso nt he evolutionary trajectory toward their tailoredp rogeny may shed some light on this aspect of the enzyme's structure-function relationship as the amount of hydroxylated product produced by the halogenases significantly drops in the course of evolution. [34,37] To day,F e/a-ketoglutarate-dependenth alogenases may already be applied for the synthesis of compounds that can be used in structure-activity relationship (SAR) studies. [63] Looking forward, the introduction of aKGHs into biosynthetic pathways, analogously to studies carried out with flavin-dependent halogenases, [47a, 64] mayb ea na ttractive tool to increaset he diversity of accessible products and to synthesize bioactive molecules or valuable intermediates.…”

“…The low activity of these enzymes (e.g.,7TTN (total turnover number)f or SyrB2, [27] see Ta ble 1) andt he mandatory covalent tethering of the substrate to the carrierp rotein has frustrated early attempts to use aKGHs for the biocatalytic CÀHf unctionalizationo fa mino acid type substrates. However,t he recent discovery of freestanding aKGHs has rekindled interesti nt he area of enzymatic C(sp 3 )-halogenation and has led to the identification [32,36] and tailoring [34,37] of aKGHs for academic and industrial applications.…”

“…CB2 was also shown to brominate 17,t houghi th ad lower preference for bromide than chloride. [37] In af urthers tudy to expand the substrate-scope of freestanding aKGHs, Hçbenreicha nd co-workers recently investigated the activitiesa nd selectivities of Hw-WelO15 from Hapalosiphon weltwitschii IC-52-3 (identical to WelO5*) and Wi-WelO15 variants towards fiven on-natural hapalindole derivatives. [34] Through comparison of substrate acceptance by the enzymes,t he authors observed that the hapalindole vinyl group was not relevant for substrate recognitionw hereas the presence of the isonitrile group was important as substrates lacking it were hardly converted.…”

Enzyme Multifunctionality by Control of Substrate Positioning Within the Catalytic Cycle—A QM/MM Study of Clavaminic Acid Synthase

“…[78] Computational studies revealed that such interactions can channel the reaction towards cleavage of the less favourable CÀHb ond,a so bserved forp rolyl 4-hydroxylase [79] and flavonol synthase, [73] or guide the order of reactions, as in the case of the ergothioneine biosynthesis enzyme EgtB. [80] The introductiono famutation within the second coordination sphere can alter the regio-and stereoselectivity of the reaction, as was demonstrated in mutagenesisstudies of the halogenase WelO5* [81] and PhrA. [22] Ta ken together,i ts eems that the key to hydroxylation is maintaining the interactions that stabilise the enzyme-substrate complex, whereas desaturation is hindered by the necessity to break favourable interactions to re-positiont he substrate-derived radicala nd provide properc ontact between the abstracted hydrogen andt he Fe III ÀOH moiety.…”