The Pseudomonas aeruginosa nirE gene encodes the S‐adenosyl‐L‐methionine‐dependent uroporphyrinogen III methyltransferase required for heme d₁ biosynthesis

Abstract

Biosynthesis of heme d1, the essential prosthetic group of the dissimilatory nitrite reductase cytochrome cd1, requires the methylation of the tetrapyrrole precursor uroporphyrinogen III at positions C‐2 and C‐7. We produced Pseudomonas aeruginosa NirE, a putative S‐adenosyl‐l‐methionine (SAM)‐dependent uroporphyrinogen III methyltransferase, as a recombinant protein in Escherichia coli and purified it to apparent homogeneity by metal chelate and gel filtration chromatography. Analytical gel filtration of purified NirE indicated that the recombinant protein is a homodimer. NirE was shown to be a SAM‐dependent uroporphyrinogen III methyltransferase that catalyzes the conversion of uroporphyrinogen III into precorrin‐2 in vivo and in vitro. A specific activity of 316.8 nmol of precorrin‐2 h−1·mg−1 of NirE was found for the conversion of uroporphyrinogen III to precorrin‐2. At high enzyme concentrations NirE catalyzed an overmethylation of uroporphyrinogen III, resulting in the formation of trimethylpyrrocorphin. Substrate inhibition was observed at uroporphyrinogen III concentrations above 17 μm. The protein did bind SAM, although not with the same avidity as reported for other SAM‐dependent uroporphyrinogen III methyltransferases involved in siroheme and cobalamin biosynthesis. A P. aeruginosa nirE transposon mutant was not complemented by native cobA encoding the SAM‐dependent uroporphyrinogen III methyltransferase involved in cobalamin formation. However, bacterial growth of the nirE mutant was observed when cobA was constitutively expressed by a complementing plasmid, underscoring the special requirement of NirE for heme d1 biosynthesis.

Molecular hijacking of siroheme for the synthesis of heme and d 1 heme

“…This operon is thought to encode all the enzymes for heme d 1 biogenesis. So far only the function of NirE has been unambiguously determined as an S-adenosylmethionine (AdoMet)-dependent uroporphyrinogen methyltransferase (11,12), a finding that is consistent with previous labeling studies that demonstrate the synthesis of d 1 heme must proceed via precorrin-2 ( Fig. 1 A and C) (13).…”

“…We believe that the oxo groups are formed by the action of NirJ, a radical AdoMet enzyme (20), and that the double bond in the propionate side chain attached to C17 may be mediated by NirF, which, being periplasmic, must catalyze the last step. We have shown that the periplasmic NirF is required for d 1 heme production whereas two other periplasmic proteins (NirC and NirN) coded for in the biogenesis operon are not (11,23). Thus we conclude that NirF catalyzes the final step in d 1 production unless the product of NirF activity is imported to the cytoplasm for further processing, an energetically expensive possibility.…”

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

“…10). Precorrin-2 is a known intermediate in the biosynthesis of siroheme (163,169,170), cobalamin (165,171), coenzyme F 430 (172), and heme d 1 (173,174) ( Fig. 10).…”

Lateral Transfer of the Denitrification Pathway Genes among Thermus thermophilus Strains

“…However, what seems to be quite unusual in the nir cluster of T. thermophilus is the absence of homologues to genes putatively implicated in the synthesis of heme d1. As an example, all of the nir clusters thus far described contain a gene (nirE) coding for uroporphyrinogen III methyltransferase (EC 2.1.1.107) (26). Its absence in the cluster of T. thermophilus could be explained by the presence of a homologue in the chromosome of T. thermophilus (TTC0308 in HB27).…”