Identification of a gene essential for protoporphyrinogen IX oxidase activity in the cyanobacterium Synechocystis sp. PCC6803

Abstract

Protoporphyrinogen oxidase (Protox) catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX during the synthesis of tetrapyrrole molecules. Protox is encoded by the hemY gene in eukaryotes and by the hemG gene in many γ-proteobacteria, including Escherichia coli. It has been suggested that other bacteria possess a yet unidentified type of Protox. To identify a unique bacterial gene encoding Protox, we first introduced the Arabidopsis hemY gene into the genome of the cyanobacterium, Synechocystis sp. PCC6803. We subsequently mutagenized the cells by transposon tagging and screened the tagged lines for mutants that were sensitive to acifluorfen, which is a specific inhibitor of the hemY-type Protox. Several cell lines containing the tagged slr1790 locus exhibited acifluorfen sensitivity. The slr1790 gene encodes a putative membrane-spanning protein that is distantly related to the M subunit of NADH dehydrogenase complex I. We attempted to disrupt this gene in the wild-type background of Synechocystis, but we were only able to obtain heteroplasmic disruptants. These cells accumulated a substantial amount of protoporphyrin IX, suggesting that the slr1790 gene is essential for growth and Protox activity of cells. We found that most cyanobacteria and many other bacteria possess slr1790 homologs. We overexpressed an slr1790 homolog of Rhodobacter sphaeroides in Escherichia coli and found that this recombinant protein possesses Protox activity in vitro. These results collectively demonstrate that slr1790 encodes a unique Protox enzyme and we propose naming the slr1790 gene “hemJ.

Discovery of a Gene Involved in a Third Bacterial Protoporphyrinogen Oxidase Activity through Comparative Genomic Analysis and Functional Complementation

“…Kato et al recently identified HemJ in Synechocystis in this fashion by introducing plant-derived acifluorfen-sensitive PPO into cells and then screening mutants for acifluorfen sensitivity (24). This approach is suited for the identification of novel enzymes where a second, well-characterized enzyme exists that catalyzes the reaction of interest and for which a novel inhibitor also exists, but it does not easily lend itself to all enzyme systems.…”

“…Kato et al (24) noted that they could not identify hemJ, hemG, or hemY in Archaea, Actinobacteria, Acidibacteria, Deinococcus, Fusobacteria, Spirochaetes, and Thermotogae and posited that these organisms may contain other, yet-to-beidentified, novel PPO enzymes. This is clearly true in Archaea, where, with limited exceptions, the organisms do not possess HemF/N, HemG/Y/J, or HemH as the terminal pathway enzymes essential for protoheme biosynthesis but instead utilize a different pathway from uroporphyrinogen to heme that involves S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase, precorrin-2 dehydrogenase, and the ahbnir genes (47).…”

“…Whether this additional component is an as-yet-unidentified protein that did not manifest itself in our genome screen or simply a redox cofactor is something that requires additional study. Interestingly, Kato et al (24) reported that a partially purified HemJmaltose binding protein fusion from Rhodobacter sphaeroides could catalyze the oxidation of protoporphyrinogen to protoporphyrin without any added cofactors. This led them to speculate that HemJ could utilize molecular oxygen for the reaction.…”

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

“…Unfortunately, relatively little is known about PgdH2. It is a membrane-bound protein but may require a soluble factor for activity (278,279). This protein has not been purified to homogeneity, and there are no data to support the presence or absence of a cofactor.…”

Tetrapyrrole Metabolism inArabidopsis thaliana

“…Currently three types of PPO have been reported: HemG (Boynton et al 2009), HemJ (Kato et al 2010), and HemY (Lermontova et al 1997;Narita et al 1996). While HemG and HemJ are both primarily of bacterial origin, HemY is found in both eukaryotes and prokaryotes.…”