title: Molecular basis for the substrate stereoselectivity in tryptophan dioxygenase.
abstract: Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are the only two heme proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine. While human IDO is able to oxidize both L- and D-Trp, human TDO (hTDO) displays major specificity for L-Trp. In this work, we aim to interrogate the molecular basis for the substrate stereoselectivity of hTDO. Our previous molecular dynamics simulation studies of Xanthomonas campestris TDO (xcTDO) showed that a hydrogen bond between T254 (T342 in hTDO) and the ammonium group of the substrate is present in the L-Trp-bound enzyme, but not in the D-Trp-bound enzyme. The fact that this is the only notable structural alteration induced by the change in the stereo structure of the substrate prompted us to produce and characterize the T342A mutant of hTDO to evaluate the structural role of T342 in controlling the substrate stereoselectivity of the enzyme. The experimental results indicate that the mutation only slightly perturbs the global structural properties of the enzyme but totally abolishes the substrate stereoselectivity. Molecular dynamics simulations of xcTDO show that T254 controls the substrate stereoselectivity of the enzyme by (i) modulating the hydrogen bonding interaction between the NH(3)(+) group and epoxide oxygen of the ferryl-indole 2,3-epoxide intermediate of the enzyme and (ii) regulating the dynamics of two active site loops, loop(250-260) and loop(117-130), critical for substrate binding.
authors.affiliation: Departamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires C1428EHA, Argentina.
authors.forename: Luciana
authors.initials: L
authors.lastname: Capece
authors.forename: Ariel
authors.initials: A
authors.lastname: Lewis-Ballester
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Marti
authors.forename: Dario A
authors.initials: DA
authors.lastname: Estrin
authors.forename: Syun-Ru
authors.initials: SR
authors.lastname: Yeh
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D055162:Biocatalysis; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013237:Stereoisomerism; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014366:Tryptophan Oxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D013912:Threonine; D014364:Tryptophan; D014366:Tryptophan Oxygenase
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