title: X-ray structure of ornithine decarboxylase from Trypanosoma brucei: the native structure and the structure in complex with alpha-difluoromethylornithine.
abstract: Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, alpha-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been determined by X-ray crystallography to 2.0 A resolution. The protein crystallizes in the space group P2(1) (a = 66.8 A, b = 154.5 A, c = 77.1 A, beta = 90.58 degrees ), with two dimers per asymmetric unit. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determined to 2.9 A resolution by molecular replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC is a beta/alpha-barrel, and the C-terminal domain of ODC is a modified Greek key beta-barrel. In comparison to structurally related alanine racemase, the two domains are rotated 27 degrees relative to each other. In addition, two of the beta-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface are formed primarily by the C-terminal domains, which interact through six aromatic rings that form stacking interactions across the domain boundary. The PLP binding site is formed by the C-termini of beta-strands and loops in the beta/alpha-barrel. In the native structure Lys69 forms a Schiff base with PLP. In both structures, the phosphate of PLP is bound between the seventh and eighth strands forming interactions with Arg277 and a Gly loop (residues 235-237). The pyridine nitrogen of PLP interacts with Glu274. DFMO forms a Schiff base with PLP and is covalently attached to Cys360. It is bound at the dimer interface and the delta-carbon amino group of DFMO is positioned between Asp361 of one subunit and Asp332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 has rotated 145 degrees toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes are observed upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas 75235, USA.
authors.forename: N V
authors.initials: NV
authors.lastname: Grishin
authors.forename: A L
authors.initials: AL
authors.lastname: Osterman
authors.forename: H B
authors.initials: HB
authors.lastname: Brooks
authors.forename: M A
authors.initials: MA
authors.lastname: Phillips
authors.forename: E J
authors.initials: EJ
authors.lastname: Goldsmith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D000518:Eflornithine / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D011993:Recombinant Fusion Proteins; D011732:Pyridoxal Phosphate; D009955:Ornithine Decarboxylase; D000518:Eflornithine