Cloning and Identification of the hemG Gene Encoding Protoporphyrinogen Oxidase (PPO) of Escherichia coli K-12

Abstract

Cells of the VSR751 strain, which was previously isolated as a photoresistant revertant of the visA-deleted (hemH-deleted) strain of Escherichia coli K-12, accumulated uroporphyrin (uro), coproporphyrin (copro) and protoporphyrin IX (proto), but did not accumulate as much protoporphyrin as cells of the parental strain (hemH-deleted). Therefore, we concluded that strain VSR751 must be defective in protoporphyrinogen oxidase (PPO), the product of the hemG gene. By complementation analysis using VSR751, we isolated and identified this gene. The hemG gene is located at 86 mim on the E. coli chromosome, just upstream of the rrnA operon, and is transcribed clockwise in the same direction as the rrnA operon. This gene encodes a 181-amino acid protein with a calculated molecular mass of about 21 kDa. Sequence analysis revealed the presence of flavodoxin motif, suggesting tha a cofactor of this enzyme is flavin mononucleotide, which is consistent with the previous report that the mammalian PPO had the flavin cofactor.

TolC-Dependent Exclusion of Porphyrins in Escherichia coli

“…Further, four molecules of porphobilinogen are condensed to form uroporphyrinogen III by the action of porphobilinogen deaminase, which is encoded by hemC, and uroporphyrinogen III synthase, which is encoded by hemD (29,35). Uroporphyrinogen III then is converted to protoporphyrin IX by successive decarboxylation and oxidation by HemE, HemF or HemN, and HemG (9,14,24,26,30,40,41). Finally, ferochelatase, encoded by hemH, converts protoporphyrin IX to heme by inserting Fe 2ϩ into protoporphyrin IX (12).…”

The cyanobacterial protoporphyrinogen oxidase HemJ is a new b-type heme protein functionally coupled with coproporphyrinogen III oxidase

“…HemJ.f bands (marked as CN1 and CN2 in Fig. 1) were tentatively assigned as dimeric (HemJ [2]) and tetrameric (HemJ [4]) HemJ.f oligomers, respectively. …”

“…1). Three analogous enzymes HemY, HemG, and lastly HemJ (recently reannotated as PgoX, PgdH1 and PgdH2 (2)), exhibiting no mutual homology, have been found to serve as a protoporphyrinogen oxidase (3)(4)(5). HemY is an oxygen-dependent enzyme of approximately 55 kDa, creating membrane bound dimers and using flavin-adenine dinucleotide as a cofactor (6).…”

Molecular Characterization and Subcellular Localization of Protoporphyrinogen Oxidase in Spinach Chloroplasts

“…The resulting plasmids, pP-pProtox (for the precursor form) and pM-pProtox (for the mature form), were introduced into the E. coli BT3 (⌬hemG::Km r ), which is obtained by replacement from the wild-type hemG gene to the hemG::Km r allele by homologous recombination (Narita et al, 1999). For a positive control, the hemG gene of E. coli (Nishimura et al, 1995a) was cloned into a pCR 2.1 vector and transformed into BT3 (⌬hemG::Km r ) cells in the same manner. Transformants were plated on Luria-Bertani (LB) plates containing 1% (w/v) bactotrypton, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, 50 g/mL ampicillin, and 25 g/mL kanamycin at 37°C.…”