CLONING, DNA-SEQUENCE, AND COMPLEMENTATION ANALYSIS OF THE SALMONELLA-TYPHIMURIUM HEMN GENE ENCODING A PUTATIVE OXYGEN-INDEPENDENT COPROPORPHYRINOGEN-III OXIDASE

Coproporphyrinogen oxidation is a late step in heme biosynthesis. The biochemically characterized eukaryotic coproporphyrinogen III oxidases have an obligate requirement for molecular oxygen, and a similar enzy me is encoded by the hemF gene in Salmonella typhimurium. Anaerobic he me synthesis requires an oxygen-independent coproporphyrinogen oxidase , which is probably encoded by the hemN gene in S. typhimurium. The he mN gene has been cloned from an insertion mutant. The nucleotide seque nce was obtained and used for PCR amplification of the wild-type gene. A single open reading frame was identified as the hemN gene on the ba sis of its interruption by the insertion mutation and plasmid compleme ntation studies of hemF hemN double mutants. The predicted HemN protei n has 38% amino acid sequence identity to a putative anaerobic Rhodoba cter sphaeroides coproporphyrinogen oxidase. The hemN RNA 5' end and t he inferred transcription initiation site were mapped by primer extens ion. The 52.8-kDa HemN protein is expressed from the second ATG codon of the hemN open reading frame. An open reading frame with an unknown function directly upstream of hemN has a striking amino acid sequence, including Il acidic residues in a row.