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J Biol Chem. 1995 Apr 7;270(14):8076-80.

Cloning of a human cDNA for protoporphyrinogen oxidase by complementation in vivo of a hemG mutant of Escherichia coli.

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Department of Biophysics, Faculty of Science, Kyoto University, Japan.


Protoporphyrinogen oxidase (PPO; EC is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway. Hemes are essential components of redox enzymes, such as cytochromes. Thus, a hemG mutant strain of Escherichia coli deficient in PPO is defective in aerobic respiration and grows poorly even in rich medium. By complementation with a human placental cDNA library, we were able to isolate a clone that enhanced the poor growth of such a hemG mutant strain. The clone encoded the gene for human PPO. Sequence analysis revealed that PPO consists of 477 amino acids with a calculated molecular mass of 50.8 kilodaltons. The deduced protein exhibited a high degree of homology over its entire length to the amino acid sequence of PPO encoded by the hemY gene of Bacillus subtilis. The NH2-terminal amino acid sequence of the deduced PPO contains a conserved amino acid sequence that forms the dinucleotide-binding site in many flavin-containing proteins. Northern blot analysis revealed the synthesis of a 1.8-kilobase pair mRNA for PPO. A homogenate of the monkey kidney COS-1 cells that had been transfected with the cDNA had much higher PPO activity than an extract of control cells, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO. Furthermore, the cDNA was expressed in vitro as 51-kilodalton protein, and after incubation with isolated mitochondria the protein was found to be located in the mitochondria, having just the same size as before, an indication that PPO is a mitochondrial enzyme and has no apparent transport-specific leader sequence.

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