Directed Evolution of a Tryptophan 2,3‐Dioxygenase for the Diastereoselective Monooxygenation of Tryptophans

Abstract

Herein, we report an engineered enzyme that can monooxygenate unprotected tryptophan into the corresponding 3a‐hydroxyhexahydropyrrolo[2,3‐b]indole‐2‐carboxylic acid (HPIC) in a single, scalable step with excellent turnover number and diastereoselectivity. Taking advantage of directed evolution, we analyzed the stepwise oxygen‐insertion mechanism of tryptophan 2,3‐dioxygenases, and transformed tryptophan 2,3‐dioxygenase from Xanthomonas campestris into a monooxygenase for oxidative cyclization of tryptophans. It was revealed that residue F51 is vital in determining the product ratio of HPIC to N′‐formylkynurenine. Our reactions and purification procedures use no organic solvents, resulting in an eco‐friendly method to prepare HPICs for further applications.

Current Advances in the Enzyme Engineering of O2‐Dependent Enzymes – Boosting the Versatility and Applicability of Oxygenases and Oxidases

“…The bulky residues form van der Waals interactions that position the tryptophan-2,3-epoxide intermediate and permit the 2 nd oxygenation; smaller polar residues prevent this reaction. [45] A new ultra-high-throughput screening method evolved the D-amino acid oxidase from Rhodotorula gracilis (RgDAAOx). The method is based on the hydrogel encapsulation of single yeast cells followed by fluorescence-activated cell sorting for screening (FACS).…”

“…A double mutant F51S/T254S showed the highest mono‐/dioxygenation ratio of 6 : 1, whereas the double mutant F51M/Q127Y showed the highest TON with 3312. The bulky residues form van der Waals interactions that position the tryptophan‐2,3‐epoxide intermediate and permit the 2 nd oxygenation; smaller polar residues prevent this reaction [45] …”

Construction of Smart Stimuli‐Responsive DNA Nanostructures for Biomedical Applications

“…To solve these problems, ah oneycomb MnO 2 (hMNS) nanosponge-sustaineda utocatalytic HCR-DNAzyme circuit was constructed for the self-sufficient DNAzyme catalytic cleavage with exponentiala mplification performance ( Figure 4D). [97] The HCR and DNAzyme were designed to synergistically cross-activate each other with increased amplification capacity.T he hMNS nanocarrier could effectively protect and deliver the DNA probes of the autocatalyticc ircuit. In addition, glutathione (GSH) disassembled the hMNS nanocarrier with the concomitantr eleaseo fD NA probesa nd Mn 2 + payloadsa sD NA-zyme cofactor.S ubsequently,t he miRNA initiated the HCRmediated assembly of DNAzymen anowires to catalyzethe efficient substrate cleavage with sufficient intracellular Mn 2 + ions.…”

“…Reproduced from ref. [97] with permission from Wiley,c opyright 2020. (E) The immunostimulatory CpG-involved HCR polymeric DNA wires and spheres for enhanced immunostimulation.…”

Synthetic studies of sumalactones A - D and Raistrickindole A

“…[102] In 2020, Wei et al developed an enzymatic system xcTDO that converted L-tryptophan into HPIC at high scale with great diastereoselectivity (Scheme 3.43). [103] Scheme 3.43: Oxidation cyclisation of tryptophan into HPIC Other methods of the indole ring oxidation into dihydroxyl compounds were also studied in great detail which inspired the final oxidation step. Kawasaki et al reported that oxidation of a protected indole with a molybdenum complex gave rise to the dihydroxylated product on the 5-membered ring.…”