Crystal structure of human uroporphyrinogen III synthase

Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin bios ynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxy methylbilane, to the macrocyclic uroporphyrino gen III, which is used in se veral different pathways to form heme, siroheme, chlorophyll, F-430 and vit amin B-12. U3S activity is essential in all organisms, and decreased activi ty in humans leads to the autosomal recessive disorder congenital erythropo etic porphyria. We have determined the crystal structure of recombinant hum an U3S at 1.85 Angstrom resolution. The protein folds into two alpha/beta d omains connected by a beta -ladder. The active site appears to be located b etween the domains, and variations in relative domain positions observed be tween crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanis ms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other h ighly conserved and titratable side chains were also mutated. In no case, h owever, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.