In the malarial parasite, pyrimidine biosynthesis provides the only route to these essential metabolites, as the parasite is unable to scavenge preformed pyrimidines (11C13). two unique co-substrate binding-sites on either side of the FMN, consistent with the observed ping-pong kinetics (6C9). Open in a separate window Plan 1 DHODH is usually a validated drug target for the treatment of human disease. The active metabolite of the arthritis drug leflunomide (e.g. A77 1726; Physique 1) is usually a potent inhibitor of human DHODH (10). In the malarial parasite, pyrimidine biosynthesis provides the only route to these essential metabolites, as the parasite is unable to scavenge preformed pyrimidines (11C13). Further, it has recently been exhibited that the main role of the mitochondrial electron transport chain in the parasite is usually to provide oxidized CoQ to serve as an electron acceptor in the flavin oxidation step of the DHODH catalytic cycle (14). TDP1 Inhibitor-1 We previously recognized a number of potent, species-selective inhibitors of DHODH by high-throughput screening, including N-(3,5-dichloro-phenyl)-2-methyl-3-nitro-benzamide (DCPMNB; Physique 1), and exhibited that these inhibitors bind to the same site as A77 1726 by mutagenesis of the binding pocket (15). The structural basis for the observed species selectivity is usually evident through comparison of the x-ray structures of the human and malarial enzymes, which show that this A77 1726 binding-site is usually highly variable in amino acid sequence between the enzymes from the two species (Physique 2) (7, 9, 16). While both human and malarial structures contain A77 1726 in this site, A77 1726 is usually a poor inhibitor of the malarial enzyme (16). We therefore propose the nomenclature species-selective inhibitor site to describe this binding pocket. Open in a separate window Physique 1 DHODH inhibitors. Open in a separate window Physique 2 Species-selective inhibitor binding site of = 7.5 Hz), 7.79C7.83 (m, 3H), 7.6 (t, 1H, = 7.8 Hz), 7.38 (d, 1H, = 1.5 Hz), 2.43 (s, 3H). MS 325.1 (M + H+). A77 1726 was synthesized as previously explained (16). Plasmid Construction and Site Directed Mutagenesis The previously explained restriction site at nucleotide 595 (full-length DHODH numbering) was eliminated using the QuickChange site-directed mutagenesis kit (Strategene) as recommended by the manufacturer, where the forward primer was 5-GAAAATATAATATATTACCCTATGATACTAGTAATGATAGTATATATGC-3 (altered base in strong). Next, and restriction sites were launched by mutagenesis at the N and C-terminus of the DHODH fragment from TDP1 Inhibitor-1 your producing plasmid was then subcloned into pET22b vector (Novagen) to generate the final expression construct (DHODH Protein Expression and Purification BL21-DE3 cells made up of the appropriate wild-type or mutant at 4C and the pellet re-suspended in 0.2 L lysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 10 mM imidazole, 5 mM 2-mercaptoethanol, 100 M FMN, 10% glycerol, and a protease inhibitor combination consisting of phenylmethylsulfonyl fluoride (200 M), leupeptin (1 g/ml), antipain (2 g/ml), benzamidine (10 g/ml), pepstatin (1 g/ml), and chymostatin (1 g/ml)). Triton X-100 (2% v/v) and lysozyme (1 mg/ml) were added, and the TDP1 Inhibitor-1 combination was stirred on ice for one hour before freezing in liquid nitrogen. DNase (0.05 mg/ml) was added to the thawed lysate, the mixture was sonicated on ice until cleared and centrifuged at 20000 at 4C. The producing supernatant was loaded onto a Ni-NTA column equilibrated in buffer A (50 mM HEPES pH 8.0, 150 mM NaCl, 20 mM imidazole, 5 mM 2-mercaptoethanol, 100 M FMN, 10% glycerol, 0.1% Triton X-100). The column was washed in buffer A until a stable baseline at A280 was reached, then bound enzyme was eluted with buffer B (50 mM HEPES pH 8.0, 150 mM NaCl, 300 mM imidazole, 5 mM 2-mercaptoethanol, 100 M FMN, 10% glycerol, 0.1% Triton X-100). Fractions made up of protein were pooled and concentrated with an Amicon Ultra centrifugal concentrating device (Amicon), Rabbit Polyclonal to CCRL1 desalted on a HiPrep 26/10 Desalting Column (Amersham Biosciences) equilibrated with enzyme assay buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100) and re-concentrated as above. Protein concentration was determined by Bradford analysis using bovine serum albumen as a standard. FMN concentration was determined by first warmth denaturing the protein to release the bound TDP1 Inhibitor-1 cofactor (purified enzyme typically diluted to 1 1 C 20 M), followed by measuring absorbance at 445 nm (445 = 12.5 mM?1 cm?1) to calculate FMN concentration (27). Circular Dichorism (CD) Spectroscopy CD spectra were recorded for wild-type and mutant enzyme samples (8 M protein) in 50 mM sodium phosphate pH 8.0..
