X-ray structure of ornithine decarboxylase from Trypanosoma brucei: The native structure and the structure in complex with alpha-difluoromethylonlithine

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent h omodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, al pha-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC, The stru cture of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been deter mined by X-ray crystallography to 2.0 Angstrom resolution. The protein crys tallizes in the space group P2(1) (a = 66.8 Angstrom, b = 154.5 Angstrom, c = 77.1 Angstrom, beta = 90.58 degrees), with two dimers per asymmetric uni t. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determine d to 2.9 Angstrom 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 bet a-barrel, In comparison to structurally related alanine racemase, the two d omains are rotated 27 degrees relative to each other. In addition, two of t he beta-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context o f the domain rotation. In ODC, the contacts in the dimer interface are form ed 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 be ta/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 2 35-237), The pyridine nitrogen of PLP interacts with Glu274, DFMO forms a S chiff base with PLP and is covalently attached to Cys360, It is bound at th e dimer interface and the delta-carbon amino group of DFMO is positioned be tween Asp361 of one subunit and Asp332 of the other, In comparison to the w ild-type uncomplexed structure, Cys-360 has rotated 145 degrees toward the active site in the DFMO-bound structure. No domain, subunit rotations, or o ther significant structural changes are observed upon ligand binding. The s tructure offers insight into the enzyme mechanism by providing details of t he enzyme/inhibitor binding site and allows for a detailed comparison betwe en the enzymes from the host and parasite which will aid in selective inhib itor design.