THE ROLE OF UROPORPHYRINOGEN III SYNTHASE STRUCTURE AND FLEXIBILITY IN THE FORM

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Uroporphyrinogen III synthase (URO-synthase), the fourth enzyme in the heme biosynthetic pathway, catalyzes the cyclization and D-ring isomerization of the linear tetrapyrrole hydroxymethylbilane (HMB), to form uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyll, and cobalamin. In the absence of URO-synthase, HMB rapidly and non-enzymatically cyclizes to form URO'gen I, the non-physiologic and pathogenic isomer that accumulates in patients with congenital erythropoietic porphyria [1]. Our objective is to characterize the mechanism by which this enzyme converts HMB specifically to URO III, avoiding URO I formation, and the role of enzyme structure in providing stereospecificity. The crystal structure of human URO-synthase has been reported at 1.85 resolution using a recombinant enzyme [2]. But efforts to map the enzyme's active site and to investigate its reaction mechanism were not successful due to the inability to co-crystallize the enzyme with a substrate analogue. Therefore, we have determined the NMR resonance assignments for human URO-synthase and used the chemical shift perturbation method and in silico docking experiments (Autodock v. 3.05), to map the active site residues in the large cleft between the enzyme's two globular domains [3]. We have now solved the 3D solution structure of this enzyme by NMR (unpublished). The NMR and crystal structures are very similar, except in the size of the cleft between the globular domains. While the crystal structure has a large open cleft, in the NMR solution structure the domains are closer together. To investigate the role of the enzyme's structure and flexibility in the conversion of HMB to URO III, we are performing molecular dynamics simulations on the enzyme complexed with the substrate, the activated substrate (an azafulvene), the spiro-pyrrolenine transition state intermediate, and the product. In our working hypothesis, the open/closed conformations of the crystal/NMR structures represent different states accessible to these ligands during the reaction mechanism, with the enzyme constraining the substrate conformational space in such a way to allow attack of the azafulvene only on the pyrrole carbon that results in the III isomer while protecting the carbon attack that would lead to the non-enzymatically formed I isomer. In order to estimate our SU requirements for a TeraGrid(tm) development account, we have determined a benchmark of 24 hours/nanosecond simulation time for our solvated protein-ligand system on a Dell PowerEdge 1950, with 8 cores at 2.66 GHz running NAMD version 2.6. We estimate requiring 10 ns simulation experiments involving complexes of open and closed forms of wild-type or site-directed mutant enzyme forms with multiple orientations of the enzyme's substrate, the predicted activated azafulvene form of the substrate, the proposed transition state intermediate, and the product. Additional simulations to calculate the free energy of binding of conformations in local minima will be performed for each ligand using free energy perturbation (FEP) methods. These experiments will require about 30,000 SU, and about 0.5 terabytes of disk storage. References 1. Anderson, K.E., The Porphyrias, in Cecil Textbook of Medicine, L. Goldman and C.J. Bennett, Editors. 2000, Philadelphia. p. 1123-1132. 2. Mathews, M.A., et al., Crystal structure of human uroporphyrinogen III synthase. Embo J, 2001. 20(21): p. 5832-9. 3. Cunha, L.F., et al., Human uroporphyrinogen III syntahse: NMR-Based mapping of the active site. Proteins: Structure, Function, and Bioinformatics, 2007. in press.