Structural basis of hereditary coproporphyria

Abstract

Hereditary coproporphyria is an autosomal dominant disorder resulting from the half-normal activity of coproporphyrinogen oxidase (CPO), a mitochondrial enzyme catalyzing the antepenultimate step in heme biosynthesis. The mechanism by which CPO catalyzes oxidative decarboxylation, in an extraordinary metal- and cofactor-independent manner, is poorly understood. Here, we report the crystal structure of human CPO at 1.58-A resolution. The structure reveals a previously uncharacterized tertiary topology comprising an unusually flat seven-stranded beta-sheet sandwiched by alpha-helices. In the biologically active dimer (K(D) = 5 x 10(-7) M), one monomer rotates relative to the second by approximately 40 degrees to create an intersubunit interface in close proximity to two independent enzymatic sites. The unexpected finding of citrate at the active site allows us to assign Ser-244, His-258, Asn-260, Arg-262, Asp-282, and Arg-332 as residues mediating substrate recognition and decarboxylation. We favor a mechanism in which oxygen serves as the immediate electron acceptor, and a substrate radical or a carbanion with substantial radical character participates in catalysis. Although several mutations in the CPO gene have been described, the molecular basis for how these alterations diminish enzyme activity is unknown. We show that deletion of residues (392-418) encoded by exon six disrupts dimerization. Conversely, harderoporphyria-causing K404E mutation precludes a type I beta-turn from retaining the substrate for the second decarboxylation cycle. Together, these findings resolve several questions regarding CPO catalysis and provide insights into hereditary coproporphyria.

Fig. 1.

CPO chemistry and sequence conservation. (A) Reaction catalyzed by CPO involves both oxidation and decarboxylation (5). CPO sequentially decarboxylates (26, 30) the propionates attached to A and B rings without affecting those on C and D rings. A hydrogen atom from the β-position of the propionate side chain also is removed at each step (31, 32). The chemical identity of the oxidation end product(s) remains to be elucidated. M = CH₃ and P = CH₂.CH₂.COO^-.(B) Sequence alignment, secondary structure, and location of HCP-causing mutations in human CPO. The first 110 amino acids are absent in the mature enzyme, for they are part of a mitochondrial targeting signal that is cleaved upon import. In the alignment (generated by using amps and alscript) red represents absolute identity over all sequences present in that part of the alignment. Database of Secondary Structure of Proteins-derived (33) secondary structural assignments are shown directly below the alignment with cylinders indicating α-helices and arrows denoting β-strands. Mutations known to cause HCP are indicated by one letter codes above the human sequence. The enzymatic activity of these variants (24, 34) are shown in blue (% relative to native enzyme). An asterisk denotes residues that affect the second decarboxylation step. Residues that make contact with citrate are indicated by diamonds. ⊗, proteolytic cleavage site.

Fig. 2.

Structure of human CPO. (A)2F_o - F_c electron density map (contoured at 1.5 σ) at 1.58 Å with a final model in place. The identity of the β-strands are shown on the right. (B) Topology diagram illustrating the organization of secondary structural elements in human CPO. Filled circles and triangles represent α-helices and β-strands, respectively. (C) Tertiary topology and quaternary structure. (D) Dimer interface.

Fig. 3.

Active site of human CPO. (A) Electrostatic potential mapped on to the molecular surface. The electropositive active site cleft is readily visible. The blue and red contours represent positive and negative potential (full saturation = 10 kT), respectively (figure was generated by using grasp; ref. 49). (B) F_o - F_c omit electron density of citrate bound at the active site. (C) Schematic illustration of the amino acid residues that make direct contact with the bound citrate. Dashed lines indicate H bonds, and nonbonded contacts are represented by an arc with spokes radiating toward the ligand atoms (figure was generated by using ligplot; ref. 50).

Fig. 4.

Catalytic mechanism. Only the first decarboxylation step is shown; the second step is expected to occur in exactly the same manner after the tricarboxylate intermediate undergoes a 90° counterclockwise rotation at the active site (ref. and D. Arigoni, personal communication).

Fig. 5.

Residues and structural regions affected by HCP mutations are shown with two different views of the structure. The polypeptide encoded by exon six is shown in cyan. Regions in blue represent mutation sites.