ISOLATION OF THE HEMF OPERON CONTAINING THE GENE FOR THE ESCHERICHIA-COLI AEROBIC COPROPORPHYRINOGEN-III OXIDASE BY IN-VIVO COMPLEMENTATIONOF A YEAST HEM13 MUTANT

Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesi s, catalyzes the oxidative decarboxylation of coproporphyrinogen III t o form protoporphyrinogen IX. Genetic and biochemical studies suggeste d the presence of two different coproporphyrinogen IU[ oxidases, one f or aerobic and one for anaerobic conditions. Here we report the clonin g of the hemF gene, encoding the aerobic coproporphyrinogen III oxidas e from Escherichia coli, by functional complementation of a Saccharomy ces cerevisiae HEM13 mutant. An open reading frame of 897 bp encoding a protein of 299 amino acids with a calculated molecular mass of 34.3 kDa was identified. Sequence comparisons revealed 43% amino acid seque nce identity with the product of the S. cerevisiae HEM13 gene and 90% identity with the product of the recently cloned Salmonella typhimuriu m hemF gene, while a structural relationship to the proposed anaerobic enzyme from Rhodobacter sphaeroides was not obvious. The hemF gene is in an operon with an upstream open reading frame (orf1) encoding a 31 .7-kDa protein with homology to an amidase involved in cell wall metab olism. The hemF gene was mapped to 52.6 min of the E. coli chromosome. Primer extension experiments revealed a strong transcription initiati on site upstream of orf1. A weak signal, possibly indicative of a seco nd promoter, was also identified just upstream of the hemF gene. A reg ion containing bent DNA (Bent 111), previously mapped to 52.6 min of t he E. coli chromosome, was discovered in the 5' region of orf1. Two po tential integration host factor binding sites were found, one close to each transcription start site. An open reading frame (orf3) transcrib ed in a direction opposite that of the hemF gene was found downstream of the hemF gene. It encodes a protein of 40.2 kDa that showed signifi cant homology to proteins of the XylS/AraC family of transcriptional r egulators.