title: Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding.
abstract: Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific heme-containing enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe (2+)) form and in complex with its substrate l-tryptophan ( l-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to l-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 A resolution, respectively, in binary complexes with l-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of l-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K d values for l-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 microM reduced), H55A TDO (11.8 microM oxidized, 3.7 microM reduced), and H55S TDO (18.4 microM oxidized, 5.3 microM reduced).
authors.affiliation: EaStCHEM, School of Chemistry, University of Edinburgh, UK.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Thackray
authors.forename: Chiara
authors.initials: C
authors.lastname: Bruckmann
authors.forename: J L Ross
authors.initials: JL
authors.lastname: Anderson
authors.forename: Laura P
authors.initials: LP
authors.lastname: Campbell
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Mowat
authors.forename: Farhad
authors.initials: F
authors.lastname: Forouhar
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Chapman
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D014366:Tryptophan Oxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016959:Xanthomonas campestris / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D006639:Histidine; D014366:Tryptophan Oxygenase