Structural and kinetic characterization of mutant human uroporphyrinogen decarboxylases

Abstract

Porphyria cutanea tarda (PCT) is caused by inhibition of uroporphyrinogen decarboxylase (URO-D) activity in hepatocytes. Subnormal URO-D activity results in accumulation and urinary excretion of uroporphyrin and heptacarboxyl porphyrin. Heterozygosity for mutations in the URO-D gene is found in the familial form of PCT (F-PCT). Over 70 mutations of URO-D have been described but very few have been characterized structurally. Here we characterize 3 mutations in the URO-D gene found in patients with F-PCT, G318R, K297N, and D306Y. Expression of the D306Y mutation results in an insoluble recombinant protein. G318R and K297N have little effect on the structure or activity of recombinant URO-D, but the proteins display reduced stability in vitro.

Figure 1. SDS-PAGE analysis of soluble and insoluble fractions from mutant URO-D expression

Mutant recombinant URO-Ds were expressed in Rosetta2(λDE3) pLysS cells (Novagen). Cells were pelleted and resuspended in 50 mM Tris pH 8.0 and 100 mM NaCl. Cells were lysed by sonication and centrifuged 45 min at 11,000 × g. The supernatants are presented as “S” (soluble fraction) and the pellets as “I” (insoluble fraction). A sample of each preparation containing 10 μg of protein was separated by 10% SDS-PAGE. Every pellet contained small amounts of recombinant URO-D. In all mutants except D306Y URO-D was present in the soluble fraction. Purified wild type URO-D (rhUroD) is shown in the far right lane as a standard. Molecular weight markers are shown in the far left lane.

Figure 2. Overlay of wild type URO-D and mutants G318R and K297N

A ribbon diagram of the wild type URO-D homodimer is shown in magenta. Monomers of G318R (slate) and K297N (lime) were aligned to wild type URO-D by least-squares overlap of all C-alpha atoms. The product of the URO-D reaction, coproporphyrinogen III (red) is shown in each of the active sites (22). The side chains R318 (blue) and K297 (green) are highlighted as space filling models. The side chain D306 (yellow) lies at the dimer interface.