title: Mutation of cysteine residues alters the heme-binding pocket of indoleamine 2,3-dioxygenase-1.
abstract: The hemoprotein indoleamine 2,3-dioxygenase-1 (IDO1) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. Interest in IDO1 continues to grow, due to the ever expanding influence IDO1 plays in the immune response. This study examined the contribution of all individual cysteine residues towards the overall catalytic properties and stability of recombinant human IDO1 via mutagenesis studies using a range of biochemical and spectroscopic techniques, including in vitro kinetic assessment, secondary structure identification via circular dichroism spectroscopy and thermal stability assessment. Upon mutation of cysteine residues we observed changes in secondary structure (principally, shifting from α-helix/β-sheet features to random coil structures) that produced out of plane heme torsion and puckering, changes to thermal stability (including gains in stability for one mutant protein) and differences in enzymatic activity (such as, increased ability to convert non-natural substrates, e.g.d-tryptophan) from wild type IDO1 enzyme.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Christopher J D
authors.initials: CJ
authors.lastname: Austin
authors.forename: Priambudi
authors.initials: P
authors.lastname: Kosim-Satyaputra
authors.forename: Jason R
authors.initials: JR
authors.lastname: Smith
authors.forename: Robert D
authors.initials: RD
authors.lastname: Willows
authors.forename: Joanne F
authors.initials: JF
authors.lastname: Jamie
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000235:genetics*; D004795:Enzyme Stability; D006418:Heme / Q000378:metabolism*; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C576710:IDO1 protein, human; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D011994:Recombinant Proteins; D006418:Heme; D003545:Cysteine
keywords: Altered substrate binding site; Cysteine residues; Indoleamine 23-dioxygenase; Kynurenine pathway; Site-directed mutagenesis; Tryptophan metabolism