A Josias, AV Corrigall, RE Kirsch, PN Meissner
Lennox Eales Porphyria Laboratories, UCT/MRC Liver Research Centre, Dept. of Medicine, University of Cape Town, South Africa
Variegate porphyria (VP) is a low penetrant autosomal dominant disorder of haem metabolism resulting from defects in protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the haem biosynthetic pathway (1). VP is highly prevalent in South Africa, the result of a founder effect, now confirmed genetically as a single point mutation (R59W), and is present in the majority of VP patients in South Africa (2,3). Missense mutations may affect substrate specificity, stability or electronic catalysis, the ability to bind and utilise the FAD cofactor or the ability to translocate to the mitochondria or the correct compartment within. As these factors may impair normal porphyrin and haem biosynthesis, their study may yield important insights into VP.
A recent study in our laboratory investigated the effect of Arg59 on catalysis and/or cofactor binding (4). The R59W founder mutation, in addition to a conservative mutation (R59K) and two non-conservative mutations (R59S and R59I) were generated, expressed, purified and partially characterised. All mutants had reduced PPOX activity but were able to bind FAD, apart from R59W in which the binding was dramatically reduced. The findings suggested that the positive charge at Arg59 is probably involved in catalysis directly and not only in FAD binding. It is speculated that the reduced FAD binding in Arg59 may be due to the bulky nature of the aromatic tryptophan. The recently solved crystal structure of the mitochondrial form of tobacco PPOX(5) revealed that Asn67(≡R59 in human) is positioned on a loop between the isoalloxazine ring of FAD and the substrate binding site. Hence, a bulky tryptophan at this position may indeed interfere with FAD and substrate binding.
Here we examine the effects of aromaticity at codon 59 by a partial characterisation of recombinant wild type and 2 R59 mutant human PPOXs. The R59F and R59Y mutant PPOXs were constructed by site-directed mutagenesis (GeneEditor kit). Wild type and mutant human PPOXs were expressed in E. coli using the pTrc-His vector and purified to homogeneity by metal affinity chromatography (Talon resin).
R59F human PPOX had a negligible specific activity (20.8±2.4nmol/mg/min) and a markedly reduced kcat(0.02±0.002sec-1)when compared to wild type (2471.4±381.6 nmol/mg/min and 2.19±0.32sec-1, respectively) and similar to that reported for the clinically relevant, R59W (4). The relatively invariant Kms for the wild type (0.92+ 0.19 µM) and R59F (1.42± 0.12 µM) suggests that the reduction in specific activity for the R59F mutant PPOX is not attributed to a reduced affinity for substrate. We speculate that the rigidity of the aromatic phenylalanine may disrupt the structural integrity of the active site. Examination of cofactor binding in wild type and R59F and R59Y mutant PPOX revealed that the presence of an aromatic residue at codon 59 alone is not sufficient to prevent FAD binding and that the degree of ‘molecular bulk’ in this position may be responsible for the severely compromised FAD binding previously reported for the R59W mutant.
References
1. Deybach J-C, Puy H, Robreau A-M, Lamoril, J, Da Silva, V, et al (1996). Mutations in the protopporphyrinogen oxidase gene in patients with porphyria. Hum Mol Genet 5:407-410.
2. Meissner PN, Dailey TA, Hift RJ, Ziman M, Corrigall AV et al (1996). A R59W defect in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria. Nat Genet 13:95-97.
3. Warnich MJ, Kotze IM, Groenewald JZ, Groenewald MG, van Brakel CJ et al (1996). Identification of the three mutations and associated haplotypes in the protoporphyrinogen oxidase gene in South African families with variegate porphyria. Hum Mol Genet 5:981-984.
4. Maneli MH, Corrigall AV, Klump HH, Davids LM, Kirsch RE et al (2003). Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases. Biochim Biophys Acta 1650 10-21.
5. Koch M, Breithhaupt C, Kiefersauer R, Freigang J, Huber R. et al.(2004). Crystal structure of protoporphyrinogen –IX oxidase: a key enzyme in haem and chlorophyll biosynthesis.