Human Uroporphyrinogen III Synthase: High Affinity Purification, NMR Resonance Assignments and Mapping of the Active Site NMR Studies Did Not Support a Heme Enzyme Complex. Uroporphyrinogen-III-synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic percursor of heme. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Previously, the crystal structure of human URO-synthase was determined. To characterize the active site of human URO-synthase and its possible interaction in a cytosolic complex with hydroxymethylbilane-synthase (HMB-synthase) or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, the three recombinant human enzymes were expressed and purified to homogeneity in high yield. Using an 800 MHz instrument with a cryoprobe, assignment of the URO-synthase backbone ¹³C resonances (100%), backbone ¹H_N resonances (99.6%), non-proline backbone ¹H_N and ¹⁵N resonances (94%), and side-chain ¹³C and ¹H_N resonances (85%) was achieved. The absence of chemical shift changes in the ¹⁵N spectrum of URO-synthase mixed with the homogeneous HMB-synthase apoenzyme or URO-decarboxylase precluded a stable cytosolic enzyme complex. In silico docking of the URO-synthase crystal structure with an inhibitor, N_D-methyl-1-formylbilane, and the product, URO'gen III, localized the enzyme's putative active site to the cleft between the enzyme's two major domains. NMR analyses of URO-synthase titrated with N_D-methyl-1-formylbilane, or URO'gen III revealed resonance perturbations of specific residues lining the cleft, confirming the in silico docking localization of the active site. These NMR studies provide the resonance assignments for human URO-synthase, exclude the interaction of the enzyme with HMB-synthase or URO-decarboxylase in a cytosolic complex, and identify the location of the enzyme's active site.