PDBsum entry 1r3q

Lyase

PDB id: 1r3q

Contents

Protein chain

357 a.a. *

Ligands

1CP

CO2

Waters ×326

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural basis for tetrapyrrole coordination by uroporphyrinogen decarboxylase.

Authors

J.D.Phillips, F.G.Whitby, J.P.Kushner, C.P.Hill.

Ref.

EMBO J, 2003, 22, 6225-6233. [DOI no: 10.1093/emboj/cdg606]

PubMed id

14633982

Abstract

Uroporphyrinogen decarboxylase (URO-D), an essential enzyme that functions in the heme biosynthetic pathway, catalyzes decarboxylation of all four acetate groups of uroporphyrinogen to form coproporphyrinogen. Here we report crystal structures of URO-D in complex with the I and III isomer coproporphyrinogen products. Crystallization required use of a novel enzymatic approach to generate the highly oxygen-sensitive porphyrinogen substrate in situ. The tetrapyrrole product adopts a domed conformation that lies against a collar of conserved hydrophobic residues and allows formation of hydrogen bonding interactions between a carboxylate oxygen atom of the invariant Asp86 residue and the pyrrole NH groups. Structural and biochemical analyses of URO-D proteins mutated at Asp86 support the conclusion that this residue makes important contributions to binding and likely promotes catalysis by stabilizing a positive charge on a reaction intermediate. The central coordination geometry of Asp86 allows the initial substrates and the various partially decarboxylated intermediates to be bound with equivalent activating interactions, and thereby explains how all four of the substrate acetate groups can be decarboxylated at the same catalytic center.

Figure 1.
Figure 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).

Figure 3.
Figure 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[ ]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[ ]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).

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2003, 22, 6225-6233) copyright 2003.