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 metaland cofactor-independent manner, is poorly understood. Here, we report the crystal structure of human CPO at 1.58-Å resolution. The structure reveals a previously uncharacterized tertiary topology comprising an unusually flat seven-stranded ␤-sheet sandwiched by ␣-helices. In the biologically active dimer (KD ‫؍‬ 5 ؋ 10 ؊7 M), one monomer rotates relative to the second by Ϸ40°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 ␤-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.coproporphyrinogen oxidase ͉ oxidative decarboxylation ͉ mitochondria ͉ x-ray crystallography T he terminal three steps of heme biosynthesis occur within the mitochondria (1, 2). First, coproporphyrinogen III is converted to protoporphyrinogen IX in the intermembrane space (3, 4) by coproporphyrinogen oxidase (CPO) (5, 6). Thus, CPO contains an unusually long (110 residues) N-terminal targeting sequence, required for its import into the mitochondria (7,8). The substrate for CPO is generated in the cytosol (9) by uroporphyrinogen decarboxylase, and the precise mechanism by which it enters the mitochondria remains to be elucidated. Second, protoporphyrinogen oxidase mediates the six electron oxidation of protoporphyrinogen to protoporphyrin IX. This enzyme is localized to the cytoplasmic side of the inner mitochondrial membrane. Third, ferrochelatase inserts the ferrous iron to generate heme within the matrix of the mitochondria. Hence, the heme biosynthetic pathway is not only partitioned between mitochondria and cytosol, but the last three enzymes are compartmentalized within the mitochondria.Partial deficiency of CPO leads to hereditary coproporphyria (HCP), an acute hepatic porphyria inherited in an autosomal dominant fashion (10-12). The disease is characterized by abdominal pain, neuropsychiatric symptoms, and͞or cutaneous photosensitivity (13). If diagnosed early, HCP can be treat...

Crystal Structure of Uroporphyrinogen Decarboxylase from Bacillus subtilis

“…An aspartate interacting with a pyrrole NH group is proposed for E. coli PBGD and yeast and human CPOX (17,18,29). The central geometry of pyrrole's coordination by an aspartate, except in hUROD, is not identified in UPMT and CPOX, due mainly to the lack of the complex structures with a substrate or product.…”

“…Anion coordination by the pyrrole nitrogen can create a cone conformation for the macrocycle (18). An aspartate interacting with a pyrrole NH group is proposed for E. coli PBGD and yeast and human CPOX (17,18,29).…”

“…In proteins, arginine residues are prime candidates. It was proposed that two arginine residues in E. coli UPMT (33), four arginine residues in human CPOX (18), and three arginine residues in human PBGD (17) might be involved in the binding of the respective substrates.…”

Computational Characterization of the Substrate-Binding Mode in Coproporphyrinogen III Oxidase

“…Deficient activity of coproporphyrinogen III oxidase causes the accumulation and non-enzymatic oxidation of coproporphyrinogen, leading to neurological disturbances and skin photosensitivity 8 . The enzyme functions as a dimer in solution, and both the human 9 and yeast 10 enzymes have been crystallized and solved at good resolutions (2.0 Å for the yeast enzyme, and 1.58 Å for the human variant). Site-directed mutagenesis experiments have shown that several conserved residues (H131 11 , as well as R135, D274 and R275…”

“…The available crystal structures [9][10] show that coproporphyrinogen III oxidase may exist in two main conformations, distinguishable due to the relative position of helixes H2 (Ser79-Lys87) and H8…”