title: Discovery of a Promiscuous Non-Heme Iron Halogenase in Ambiguine Alkaloid Biogenesis: Implication for an Evolvable Enzyme Family for Late-Stage Halogenation of Aliphatic Carbons in Small Molecules.
abstract: The elucidation of enigmatic enzymatic chlorination timing in ambiguine indole alkaloid biogenesis led to the discovery and characterization of AmbO5 protein as a promiscuous non-heme iron aliphatic halogenase. AmbO5 was shown capable of selectively modifying seven structurally distinct ambiguine, fischerindole and hapalindole alkaloids with chlorine via late-stage aliphatic C-H group functionalization. Cross-comparison of AmbO5 with a previously characterized aliphatic halogenase homolog WelO5 that has a restricted substrate scope led to the identification of a C-terminal sequence motif important for substrate tolerance and specificity. Mutagenesis of 18 residues of WelO5 within the identified sequence motif led to a functional mutant with an expanded substrate scope identical to AmbO5, but an altered substrate specificity from the wild-type enzymes. These observations collectively provide evidence on the evolvable nature of AmbO5/WelO5 enzyme duo in the context of hapalindole-type alkaloid biogenesis and implicate their promise for the future development of designer biocatalysis for the selective late-stage modification of unactivated aliphatic carbon centers in small molecules with halogens.
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Hillwig
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Kuljira
authors.initials: K
authors.lastname: Ittiamornkul
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA. xinyuliu@pitt.edu.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Liu
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis* / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D002244:Carbon / Q000737:chemistry / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology; D054879:Halogenation; D007211:Indoles / Q000737:chemistry; D010088:Oxidoreductases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000470:Alkaloids; D001426:Bacterial Proteins; D007211:Indoles; D002244:Carbon; C030374:indole; D010088:Oxidoreductases
keywords: C−H activation; alkaloid biogenesis; evolution; halogenase; non-heme iron enzyme