Structural insight into human variegate porphyria disease

Abstract

Human protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein, converts protoporphyrinogen IX to protoporphyrin IX in the heme biosynthetic pathway. Mutations in the hPPO gene cause the inherited human disease variegate porphyria (VP). In this study, we report the crystal structure of hPPO in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of 1.9 Å. The structural and biochemical analyses revealed the molecular details of FAD and acifluorfen binding to hPPO as well as the interactions of the substrate with hPPO. Structural analysis and gel chromatography indicated that hPPO is a monomer rather than a homodimer in vitro. The founder-effect mutation R59W in VP patients is most likely caused by a severe electrostatic hindrance in the hydrophilic binding pocket involving the bulky, hydrophobic indolyl ring of the tryptophan. Forty-seven VP-causing mutations were purified by chromatography and kinetically characterized in vitro. The effect of each mutation was demonstrated in the high-resolution crystal structure.

Functional Characterization of Five Protoporphyrinogen oxidase Missense Mutations Found in Argentinean Variegate Porphyria Patients

“…In consequence, the mutations studied are most likely the cause of the reduced PPOX activities observed in patients carrying these defects. Moreover, Qin et al (2011) also found minimal or no residual activity for c.670T>G (p.W224G) and c.995G>C (p.G332A) mutations. No correlation was observed between these functional consequences and the expression or features of the disease in the patients carrying the respective mutations.…”

“…Three of these mutations c.101A>T (p.E34V), c.995G>C (p.G332A), and c.1265A>G (p.Y422C), affect amino acids highly conserved throughout evolution, whereas the mutations c.227C>T (p.S76F) and c.670T>G (p.W224G) change less conserved residues, but preserved in different mammals species such as Macaca fascicularis (monkey), Bos taurus (bovine), Sus scrofa (pig), Mus musculus (mouse), and Rattus novergicus (rat) (Protein Knowledgebase (UniProtKB), http://www.uniprot.org). In the crystal structure of human PPOX, amino acids E34, S76, W224, and Y422 are located in the FAD-binding domain, and G332 resides in the substrate-binding domain (Qin et al 2011). Besides, residues E34 and G332 are involved in the binding to cofactor and substrate, respectively (Qin et al 2011).…”

“…In the crystal structure of human PPOX, amino acids E34, S76, W224, and Y422 are located in the FAD-binding domain, and G332 resides in the substrate-binding domain (Qin et al 2011). Besides, residues E34 and G332 are involved in the binding to cofactor and substrate, respectively (Qin et al 2011). Moreover, tryptophan at position 224 is located in the hydrophobic core and so this mutation confers a hydrophilic side chain probably destabilizing the core and affecting the stability of the domain conformation (Qin et al 2011).…”

“…Besides, residues E34 and G332 are involved in the binding to cofactor and substrate, respectively (Qin et al 2011). Moreover, tryptophan at position 224 is located in the hydrophobic core and so this mutation confers a hydrophilic side chain probably destabilizing the core and affecting the stability of the domain conformation (Qin et al 2011). It would likely also be that this residue could be in the internal mitochondrial targeting signal (Morgan et al 2004;Davids et al 2006).…”

“…These mutations included 70 missense, 22 splicing defects, 29 small deletions, 15 small insertions, 1 small indels, 1 gross insertion, and 1 duplication. In several of the missense mutations, their functional effect on protein activity has been studied using in vitro expression systems Qin et al 2011). Most patients are heterozygous, exhibiting approximately 50% reduced PPOX activity (Brenner and Bloomer 1980;Deybach et al 1981).…”