Structure and mechanistic implications of a uroporphyrinogen III synthase-product complex

Abstract

Uroporphyrinogen III synthase (U3S) catalyzes the asymmetrical cyclization of a linear tetrapyrrole to form the physiologically relevant uroporphyrinogen III (uro'gen III) isomer during heme biosynthesis. Here, we report four apoenzyme and one product complex crystal structures of the Thermus thermophilus (HB27) U3S protein. The overlay of eight crystallographically unique U3S molecules reveals a huge range of conformational flexibility, including a "closed" product complex. The product, uro'gen III, binds between the two domains and is held in place by a network of hydrogen bonds between the product's side chain carboxylates and the protein's main chain amides. Interactions of the product A and B ring carboxylate side chains with both structural domains of U3S appear to dictate the relative orientation of the domains in the closed enzyme conformation and likely remain intact during catalysis. The product C and D rings are less constrained in the structure, consistent with the conformational changes required for the catalytic cyclization with inversion of D ring orientation. A conserved tyrosine residue is potentially positioned to facilitate loss of a hydroxyl from the substrate to initiate the catalytic reaction.

Figure 1

The proposed mechanism of U3S catalyzed formation of uro’gen III (3). This figure resembles that of Roessner et al. (33). Spirolactam, a transition state mimic, has been shown to be a competitive inhibitor of U3S (36), and the structure of a synthetic precursor, methylated dinitrilepyrrolenine, has been determined (6). A = CH₂CO₂, P = CH₂CH₂CO₂.

Figure 2

Crystal structures of U3S: (A) human (PDB code, 1JR2); (B, F, G, H) four apo T. thermophilus (HB27) U3S structures; (C, E) T. thermophilus (HB8) apo-U3S (PDB code 1WCW and 1WD7); (D) T. thermophilus (HB27) U3S bound to product, uro’gen III. (I) Overlap of all these U3S structures on domain 1 reveals substantial flexibility of the linker region and relative motion of the second domain. Structures have been positioned in the figure according to the angular displacement of the domain 2 relative to the most extended structure of human U3S.

Figure 3

U3S:uro’gen III product complex. (A) Final 2F_o – F_c electron density surrounding the uro’gen III ligand and nearby active site residues. (B) Stereo representation of U3S active site and bound uro’gen III. The acetate and propionate side chains of the four pyrrole rings (A, B, C and D) form hydrogen bonds to main chain amides, water molecules and two nonconserved side chains.

Figure 4

Domain closure upon ligand binding. (A) Surface representation of human apo U3S, which adopts the most open conformation observed in the various crystal structures. (B) Product bound T. thermophilus U3S showing how the domains close around the uro’gen ligand (purple). Both panels are in the same orientation as Figure 2.

Figure 5

Comparative modeling. (A) Overlap of the individual domains and linker region of the human U3S (cyan) on the TtU3S structure (green) reveals the alternate side chain position of a conserved tyrosine residue (TtU3S Tyr155, human Tyr168) that may function in removal of the C20 hydroxyl of the substrate HMB. (B) Stereo representation of uro’gen III and a modeled spiropyrollenine transition state in the active site of U3S. Rings A and B pack against the U3S protein, and rings C and especially D are more open to solvent. The modeled spiro-intermediate macrocycle is smaller in diameter and easily fits into the active site with minimal changes. Close approach between the D ring side chains and Val69 may determine the pucker of the intermediate.