Molecular phylogeny and intricate evolutionary history of the three isofunctional enzymes involved in the oxidation of protoporphyrinogen IX

Abstract

Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis.

Keywords: HemG; HemJ; HemY; heme; protoporphyrin IX; tetrapyrrole.

Fig. 1.—

Phylogenetic tree of HemG. BI tree of HemG proteins with confidence support values obtained with BI/ML/PB/RA/NJ methods shown at each branch. Only BI score is shown in the interior of branches for visibility. Distance scale is indicated by the bar on top. The cluster numbers refer to those in the Gclust 2010 data set. Cluster 26351 is an outgroup consisting of flavodoxins.

Fig. 2.—

Synteny in the hemG/hemJ replacement region in Prochlorococcus. (A) A 16S–23S-based phylogenetic tree of the Synechococcus–Prochlorococcus lineage along with deeply branched Gloeobacter violaceus. (B) Arrangement of genes in the regions containing hemG or hemJ in related Prochlorococcus strains.

Fig. 3.—

Phylogenetic tree of HemJ. BI tree of HemJ proteins with confidence support values obtained with BI/ML/PB/RA/NJ methods shown at each branch. Only a BI score is shown in the interior of branches for visibility. For multiple branches, only a BI score is assigned. Distance scale is indicated by the bar on top. The cluster numbers refer to those in the Gclust 2010 data set. Cluster 1819 contains HemJ proteins that were not yet characterized at the time of preparing the Gclust database. Clusters 40856 and 28730 are supposed to be outgroups having unknown function(s).

Fig. 4.—

Phylogenetic tree of HemY. BI tree of HemY with confidence support values obtained with BI/ML/PB/RA/NJ methods shown at each branch. Only a BI score is shown in the interior of branches for visibility. Distance scale is indicated by the bar on top. The cluster numbers refer to those in the Gclust 2010 data set. Clusters 8156 and 3943 are mixed in the major HemY group. Land plants contain both PPO1 and PPO2. Cluster 15732 contains proteins that are probably not HemY. Cluster 9674 is an outgroup consisting of amine oxidases. Note that some proteins are erroneously annotated as HemG in the original databases.

Fig. 5.—

Phylogenic trees used for AU testing. Twenty representative HemY proteins were selected, and the most likely trees were evaluated by AU test. Trees 1–15 were selected by the protml program. Trees 16–19 were tested as constrained trees. The results of the AU test are in table 3. Both JTT and WAG models were tested. Trees 1–15 should not be rejected as nonsignificant, whereas trees 16–19 were clearly abandoned. For names of eukaryotes, see table 1. Other names are the followings: Bsu, Bacillus subtilis; Caur, Chloroflexus aurantiacus; Fal, Frankia alni; Glv, Gloeobacter violaceus; Roca, Roseiflexus castenholzii; Sthe, Sphaerobacter thermophilus; Tel, Thermosynechococcus elongatus; Ter, Trichodesmium erythraeum; Tth, Thermus thermophilus; YelA, Cyanobacterium Yellowstone A-prime.

Fig. 6.—

Comparison of the structures of various HemY proteins. Structures of various HemY proteins (shown on left and center) were estimated by homology modeling with Nicotiana tabacum PPO2, Bacillus subtilis HemY, and Myxococcus xanthus HemY. Characteristic structures are annotated with numbers, which correspond to the annotations in supplementary figure S7, Supplementary Material online. The rainbow color gradation from blue to red indicates direction from the N-terminus to C-terminus.