Cloning and expression of a Porphyromonas gingivalis gene for protoporphyrinogen oxidase by complementation of a hemG mutant of Escherichia coli.
Porphyromonas gingivalis, a bacterium implicated in periodontal pathogenesis, has a growth requirement for iron protoporphyrin IX. By complementation with a P. gingivalis 381 chromosomal DNA library, we were able to isolate a clone that enhanced the poor growth of a hemG mutant of Escherichia coli. The DNA sequence analysis of this clone revealed three open reading frames (ORFs). ORF3 encoded a protein of 466 amino acids with a calculated molecular weight of 51 695 Da. The deduced amino acid sequence of the ORF3 gene had significant similarity to sequences of protoporphyrinogen oxidase (PPO) from Myxococcus xanthus (30% identical residues). When the ORF3 gene was overexpressed in E. coli, the extract had much higher PPO activity than a control extract, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO. Thus, ORF3 was named PgHemG. Furthermore, several porphyrin-related genes, including hemD, hemN and hemH, were identified in the data bases on the websites available on-line. We postulated that a porphyrin biosynthetic pathway to heme from preuroporphyrin may be conserved in P. gingivalis.
D000595:Amino Acid Sequence / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D003304:Coproporphyrinogen Oxidase; D004269:DNA, Bacterial / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D006418:Heme / Q000235:genetics; D006801:Humans; D007501:Iron / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009579:Nitrobenzoates / Q000494:pharmacology; D016366:Open Reading Frames / Q000235:genetics; D010088:Oxidoreductases / Q000187:drug effects / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D050768:Protoporphyrinogen Oxidase; D011524:Protoporphyrins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial; D014578:Uroporphyrins / Q000235:genetics
D001426:Bacterial Proteins; D003304:Coproporphyrinogen Oxidase; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D006418:Heme; D007501:Iron; D009579:Nitrobenzoates; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011498:Protein Precursors; D050768:Protoporphyrinogen Oxidase; D011524:Protoporphyrins; D014578:Uroporphyrins; C448299:iron protoporphyrin IX; C109194:HemN protein, Bacteria; C493977:HemG protein, E coli; C018425:acifluorfen