Oxygen-dependent coproporphyrinogen-III oxidase fromEscherichia coli: one-step purification and biochemical characterisation

Coproporphyrinogen-III oxidase (CPO) catalyses the conversion of coproporphyrinogen-III to protoporphyrinogen-IX in the haem biosynthetic pathway, and its deficient activity is associated with human hereditary coproporphyria. The 47% sequence identity between the oxygen-dependent CPO from Escherichia coli and its human counterpart makes the bacterial enzyme a good model system for structural studies of this disease. Therefore, we overexpressed and purified to homogeneity the oxygen-dependent CPO from E. coli and its selenomethionine derivative fused with a His6-tag. Both preparations showed a specific activity of 37 500 U mg−1, had a subunit molecular mass of 35 kDa and behaved as a compact shaped dimer. First crystallisation trials produced plate-shaped diffracting crystals