title: Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme.
abstract: Many peroxy-containing secondary metabolites have been isolated and shown to provide beneficial effects to human health. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Wupeng
authors.initials: W
authors.lastname: Yan
authors.affiliation: Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Heng
authors.initials: H
authors.lastname: Song
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Fuhang
authors.initials: F
authors.lastname: Song
authors.affiliation: Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Yisong
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Cheng-Hsuan
authors.initials: CH
authors.lastname: Wu
authors.affiliation: Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Ampon Sae
authors.initials: AS
authors.lastname: Her
authors.affiliation: Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.|Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Pu
authors.affiliation: Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Nathchar
authors.initials: N
authors.lastname: Naowarojna
authors.affiliation: Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Weitz
authors.affiliation: Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hendrich
authors.affiliation: Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.|Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Costello
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Lixin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Pinghua
authors.initials: P
authors.lastname: Liu
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USA.|Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Yan Jessie
authors.initials: YJ
authors.lastname: Zhang
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology*; D001665:Binding Sites; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme; D006900:Hydroxylation; D007211:Indoles / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011449:Prostaglandin Endoperoxides / Q000096:biosynthesis*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D007211:Indoles; D007656:Ketoglutaric Acids; D011449:Prostaglandin Endoperoxides; D014443:Tyrosine; D006418:Heme; D007501:Iron; C039286:fumitremorgin B; D010100:Oxygen