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Functional Diversity of HemN-like Proteins.

ABSTRACT: HemN is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the anaerobic oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, a key intermediate in heme biosynthesis. Proteins homologous to HemN (HemN-like proteins) are widespread in both prokaryotes and eukaryotes. Although these proteins are in most cases annotated as anaerobic coproporphyrinogen III oxidases (CPOs) in the public database, many of them are actually not CPOs but have diverse functions such as methyltransferases, cyclopropanases, heme chaperones, to name a few. This Perspective discusses the recent advances in the understanding of HemN-like proteins, and particular focus is placed on the diverse chemistries and functions of this growing protein family.

SUBMITTER: Cheng J

PROVIDER: S-EPMC10114718 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Functional Diversity of HemN-like Proteins.

Cheng Jinduo J Liu Wan-Qiu WQ Zhu Xiaoyu X Zhang Qi Q

ACS bio & med chem Au 20220118 2

HemN is a radical <i>S</i>-adenosylmethionine (SAM) enzyme that catalyzes the anaerobic oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, a key intermediate in heme biosynthesis. Proteins homologous to HemN (HemN-like proteins) are widespread in both prokaryotes and eukaryotes. Although these proteins are in most cases annotated as anaerobic coproporphyrinogen III oxidases (CPOs) in the public database, many of them are actually not CPOs but have diverse funct ...[more]

PMID: 37101745

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Project description:Previously, we screened the symbiotic gene region of the Bradyrhizobium japonicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472-1480, 2000). Here we report more details on one of the genes identified, a hemN-like gene (now called hemN(1)) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum possesses a second hemN-like gene (hemN(2)), which was then cloned by using hemN(1) as a probe. The hemN(2) gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN(1) and HemN(2), respectively) and share 53% identical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (> or = 20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK(2). In addition, maximal anaerobic hemN(1) expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the hemN(1) mutant, strains lacking a functional hemN(2) gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several c-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that B. japonicum hemN(2), but not hemN(1), encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a Salmonella enterica serovar Typhimurium hemF hemN mutant with hemN(2) only.

Project description:Previous characterization of Bacillus subtilis hemN, encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation, indicated the presence of a second hemN-like gene (B. Hippler, G. Homuth, T. Hoffmann, C. Hungerer, W. Schumann, and D. Jahn, J. Bacteriol. 179:7181-7185, 1997). The corresponding hemZ gene was found to be split into the two potential open reading frames yhaV and yhaW by a sequencing error of the genome sequencing project. The hemZ gene, encoding a 501-amino-acid protein with a calculated molecular mass of 57,533 Da, complemented a Salmonella typhimurium hemF hemN double mutant under aerobic and anaerobic growth conditions. A B. subtilis hemZ mutant accumulated coproporphyrinogen III under anaerobic growth conditions. A hemN hemZ double mutant exhibited normal aerobic and anaerobic growth, indicating the presence of a third alternative oxygen-independent enzymatic system for coproporphyrinogen III oxidation. The hemY gene, encoding oxygen-dependent protoporphyrinogen IX oxidase with coproporphyrinogen III oxidase side activity, did not significantly contribute to this newly identified system. Growth behavior of hemY mutants revealed the presence of an oxygen-independent protoporphyrinogen IX oxidase in B. subtilis. A monocistronic hemZ mRNA, starting 31 bp upstream of the translational start codon, was detected. Reporter gene fusions of hemZ and hemN demonstrated a fivefold anaerobic induction of both genes under nitrate ammonifying growth conditions. No anaerobic induction was observed for fermentatively growing B. subtilis. The B. subtilis redox regulatory systems encoded by resDE, fnr, and ywiD were indispensable for the observed transcriptional induction. A redox regulation cascade proceeding from an unknown sensor via resDE, through fnr and ywiD to hemN/hemZ, is suggested for the observed coregulation of heme biosynthesis and the anaerobic respiratory energy metabolism. Finally, only hemZ was found to be fivefold induced by the presence of H(2)O(2), indicating further coregulation of heme biosynthesis with the formation of the tetrapyrrole enzyme catalase.

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Functional Diversity of HemN-like Proteins.

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Functional Diversity of HemN-like Proteins.

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