Cloning of a human cDNA for protoporphyrinogen oxidase by complementation in vivo of a hemG mutant of Escherichia coli.
Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) 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.
Nishimura
K
K
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Taketani
S
S
Inokuchi
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005420:Flavoproteins; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D005420:Flavoproteins; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493977:HemG protein, E coli; C493978:PPOX protein, human; D050768:Protoporphyrinogen Oxidase