Fig. 1. Reaction catalyzed by URO-D and the two preceding steps in the heme biosynthesis pathway. Acetate, propionate and methyl side-chains are denoted A, P and M, respectively. Porphobilinogen deaminase (PBG-D), uroporphyrinogen III synthase (U3S). In the absence of U3S, hydroxymethylbilane cyclizes without inversion of the D-ring, to form uroporphyrinogen I, which differs from the III-isomer shown here by having an identical arrangement of A/P substituents on all four pyrrole rings. Figures 1 and 4 were generated using ChemDraw Pro (CambridgeSoft Corp., Cambridge, MA).

Fig. 2. Structure of the URO-D–product complexes. (A) Stereoview ribbon representation of the dimer, viewed down the 2-fold axis between adjacent monomers. Monomer 1 is blue with an orange beta barrel and a purple flexible loop and flanking beta strands. Monomer 2 is yellow. Coproporphyrinogen-III product is green. Coproporphyrinogen-I (not shown) overlaps almost exactly with the III-isomer. This figure was generated using Molscript (Kraulis, 1991) and Raster3D (Merritt and Bacon, 1997). (B) Sequence of human URO-D. The additional 21 residues, including a 10-histidine tag, that were inserted before Met1 are not shown explicitly in the figure, although they are present in all the proteins used for structure determination and activity assays performed in this study. The secondary structure shown above has the same color scheme as (A). Disordered residues (at N- and C-termini) are indicated with a thin line. Residues that are invariant in an alignment of 39 sequences (data not shown) are shown on a yellow background. Asp86 is indicated with a red triangle. Residues that contact the product ligand, i.e. have at least one atom within 4.0 Å of the product, are indicated below according to the color scheme indicated. (C) Electron density around the product. Stereoview of the sigmaA-weighted 2F_o–F_c map shown contoured at 1 × r.m.s.d. around the I-isomer product bound to wild-type URO-D. (D) Electrostatic potential surface at the URO-D active site. Stereoview colored on a red/white/blue scale of –0.3/0/+0.3 kT. Coproporphyrinogen-III is shown in green. Hydrogen bonds between the tetrapyrrole NH groups and Asp Oδ1 are indicated with dashed purple lines. (C) and (D) were generated using SwissPDBviewer (http://www.expasy.org/spdbv/) (Guex and Peitsch, 1997).

Fig. 3. Interactions at the active site. (A) Uroporphyrinogen I product (green), wild-type URO-D (yellow). Protein residues are shown if one atom from the residue lies within 4.0 Å of the product in at least one of the structures. Also shown is Leu88. The pyrrole rings are denoted A, B, C and D, with the D-ring being the site where acetate and propionate groups are reversed in the III-isomer product. Van der Waals’ surfaces are shown around the protein atoms, with residues nearer the viewer given a more transparent surface. Hydrogen bonds are indicated with dashed lines. The apparent hydrogen bond seen between Ala39 O and the C-ring propionate indicates that this carboxylate is protonated. (B) Same as (A), but for the III-isomer product complex. The I- and III-isomer products superimpose very closely following overlap on the protein C_α atoms. The major differences are the conformations of Arg37 and Arg 41 side chains. (C) Comparison of product bound to various URO-D variants. Structures were superimposed by overlap on the protein C_α atoms. The water molecules that lie roughly in the position of the wild-type Asp86 side chains are shown explicitly. Color scheme is indicated and is the same as in Figure 2B. This figure was generated using Molscript (Kraulis, 1991) and Raster3D (Merritt and Bacon, 1997).

Fig. 4. Proposed reaction pathway. The second figure in the series shows how the positive charge from protonation can be delocalized to the pyrrole nitrogen and explains how Asp86 promotes formation of the proposed protonated intermediate. The positively charged intermediate serves as an electron sink for release of the acetate carboxylate as carbon dioxide. Two protons are introduced in this proposed pathway, in the first and third panels.