RadH: A Versatile Halogenase for Integration into Synthetic Pathways

Abstract

Flavin-dependent halogenases are useful enzymes for providing halogenated molecules with improved biological activity,o ri ntermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate ar ange of bioactive aromatic scaffolds.S ite-directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases.Ahigh-throughput fluorescence screen was also developed, whichenabled aRadH mutant to be evolved with improved properties.Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode an ew pathwayt og enerate an ovel chlorinated coumarin "non-natural" product in E. coli.

A Perspective on Synthetic Biology in Drug Discovery and Development—Current Impact and Future Opportunities

“…(D) Engineered enzyme cascade including tyrosine ammonia-lyase (TAL), 4-coumaryl-CoA ligase (4CL), feruloyl CoA 6′-hydroxylase (F6′H), and flavin-dependent halogenase (RadH). 244 (E) Fluorinase (Fl ase ) and improved variants replace Cl with 18 F in 5′-chloro-5′-deoxyadenosine (5′-ClDA), even when the C2 position of the adenine ring presents a long moiety (brown) attached to a bioactive molecule (e.g., peptide with affinity for cancer cells). 249,251 Crystal structures of P450, UPO, catechol-O-methyltransferase (COMT), WelO5, RebH, and Fl ase correspond to PDB IDs 1JPZ, 2YOR, 1VID, 5IQS, 2OAL, and 1RQP, respectively.…”

“…4D ). 244 While flavin-dependent halogenases act on electron-rich substrates, Fe II /2OG-dependent halogenases catalyze the halogenation of unactivated aliphatic sp 3 carbon centers ( Fig. 4C ).…”

“…(C) Martinelline-derived fragment (top) and lysine (bottom), chlorinated by WelO5* variants246 and BesD,227 respectively. (D) Engineered enzyme cascade including tyrosine ammonia-lyase (TAL), 4-coumaryl-CoA ligase (4CL), feruloyl CoA 6′-hydroxylase (F6′H), and flavin-dependent halogenase (RadH) 244. (E) Fluorinase (Fl ase ) and improved variants replace Cl with18 F in 5′-chloro-5′-deoxyadenosine (5′-ClDA), even when the C2 position of the adenine ring presents a long moiety (brown) attached to a bioactive molecule (e.g., peptide with affinity for cancer cells) 249,251.…”

A Promiscuous Halogenase for the Derivatization of Flavonoids

“…The resulting intermediate known as the Wheland complex is characterized by a positive charge. In the case of RebH and other common FAD-dependent halogenases, this complex is stabilized by an interaction with aspartic or glutamic acid residues, which deprotonate the Wheland complex at the halogenated position [29,54]. This restores the aromaticity of the halogenated ring of the substrate to complete the reaction.…”

“…The well characterized halogenase RebH is for example responsible for the regiospecific halogenation of tryptophan precursor in the biosynthesis of the natural compound rebeccamycin [28]. It has been further demonstrated for the fungal flavindependent halogenase RadH that several phenolic compounds were accepted as substrates and the relaxed substrate specificity allowed the construction of artificial biosynthetic pathways [29]. Of great interest is therefore the identification of other promiscuous halogenases for the modification of a variety of natural products.…”