STRUCTURAL BASIS FOR TETRAPYROLE COORDINATION BY UROPORPHYRINOGEN DECARBOXYLASE

Uroporphyrinogen decarboxylase (URO-D) removes a carboxyl group from each of the 4 acetate side chains of uroporphyrinogen (uro'gen) without the need for cofactors to act as proton or electron donors. Subnormal activity of URO-D is the cause of porphyria cutanea tarda (PCT), the most common of the human porphyrias. Approximately 1/3 of patients with PCT are heterozygous for inherited mutations at the URO-D locus, transmitted as an autosomal dominant trait (familial-PCT). We have characterized the structural and functional effects of many of the mutations found in familial-PCT (Blood. 2001; 98:3179-3185). The enzyme is dimeric with a two fold symmetry that lies between the active sites. We have crystallized URO-D under anaerobic conditions in the presence of its fully reduced porphyrinogen substrate. Using the structure of the enzyme product complex we have begun to probe the enzyme mechanism through site-directed mutagenesis. Recombinant His-tagged human URO-D (rhURO-D) was concentrated to approximately 10 mg/ml in buffer with 10% glycerol and crystallized in the presence of enzymatically generated uro'gen I or III in an anaerobic chamber. The structure of the rhURO-D-porphyrinogen complex revealed coproporphyrinogen (copro'gen) in the active site, indicating that the uro'gen substrate had been decarboxylated but the reaction product was not released. The structure revealed no change in the configuration of URO-D compared to the unliganded protein. Copro'gen in the active site was in a domed shape with the 4 pyrrole nitrogens pulled above the plane of the macro cycle through hydrogen bonding to the carboxyl group of Asp86. These data explain why only reduced porphyrinogens can serve as substrates of URO-D as the oxidized porphyrin lacks the flexibility to adopt the domed conformation to orient the macro cycle for catalysis. Three arginine (residues 37, 41 and 50) stabilize the domed macro cycle through hydrogen bonding with the propionate side chains of the porphyrinogen. A hydrophobic domain within the active site, interacting with one quadrant of the macro cycle, is essential for decarboxylation. Mutating either of 2 phenylalanines (residues 154 and 217) to less hydrophobic residues effectively ablated decarboxylation. No residue with the potential to protonate the macro cycle is appropriately positioned within the hydrophobic domain but a domain interacting with the adjacent quadrant contains Tyr164 and Ser219, both capable of protonating the macro cycle. Mutation of either of these residues severely reduces enzymatic activity. Collectively, these data indicate that catalysis requires stabilization of the porphyrinogen substrate in a domed configuration, protonation of the macro cycle and a critical interaction between the hydrophobic domain and an acetate carboxyl group. The specific activity of hepatic URO-D is markedly reduced in humans with PCT and in experimental models of PCT (PNAS. 2001;98, 259-264), strongly suggesting that an inhibitor of URO-D is involved in the pathogenesis of the disease. Defining the structural requirements for URO-D activity may lead to the mechanism by which an inhibitor reduces enzymatic activity.