Tetrapyrrole synthesis of photosynthetic chromerids is likely homologous to the unusual pathway of apicomplexan parasites

Abstract

Most photosynthetic eukaryotes synthesize both heme and chlorophyll via a common tetrapyrrole biosynthetic pathway starting from glutamate. This pathway was derived mainly from cyanobacterial predecessor of the plastid and differs from the heme synthesis of the plastid-lacking eukaryotes. Here, we show that the coral-associated alveolate Chromera velia, the closest known photosynthetic relative to Apicomplexa, possesses a tetrapyrrole pathway that is homologous to the unusual pathway of apicomplexan parasites. We also demonstrate that, unlike other eukaryotic phototrophs, Chromera synthesizes chlorophyll from glycine and succinyl-CoA rather than glutamate. Our data shed light on the evolution of the heme biosynthesis in parasitic Apicomplexa and photosynthesis-related biochemical processes in their ancestors.

Figure 1.

Phylogenetic Trees of Ferrochelatase and ALA-Synthase. Bayesian phylogenetic trees as inferred from ALAS (A) and ferrochelatase (B) amino acid sequences, and the enzymes of the first and last step of the heme synthesis. Numbers above branches indicate Bayesian posterior probabilities. The trees demonstrate mitochondrial origin of ALAS (A) and uncovered enigmatic origins of the apicomplexan ferrochelatase (B). [See online article for color version of this figure.]

Figure 2.

Bayesian Phylogenetic Tree as Inferred from UROD Amino Acid Sequences. The tree well demonstrates the complex symbiotic origin of C. velia. Particular UROD enzymes appear to originate from cyanobacteria, the primary host nucleus (nucleus of a eukaryotic alga involved in secondary endosymbiosis), and secondary host nucleus (nucleus of a heterotrophic eukaryote that engulfed the alga). Numbers above branches indicate Bayesian posterior probabilities. [See online article for color version of this figure.]

Figure 3.

Tetrapyrrole Biosynthetic Pathways of Various Eukaryotes. Distribution of different types of the tetrapyrrole biosynthetic pathway (B) among chromalveolates (A). (1) Canonical C4 pathway of heterotrophic eukaryotes; localization differs between animal cells (Dailey et al., 2005) and yeast (Camadro et al. 1986). (2) Canonical C5 pathway that is shared by most of the photosynthetic eukaryotes. (3) Tetrapyrrole pathway of C. velia with putative localizations based on in silico predictions using SignalP and TargetP, respectively (Emanuelsson et al., 2007). (4) Heme biosynthesis of Apicomplexa; localization differs between P. falciparum and Toxoplasma gondii (Sato et al., 2004; Nagaraj et al., 2009a, 2009b, 2010a, 2010b; van Dooren et al., 2006; Wu, 2006; Shanmugam et al., 2010). Numbers of the individual steps of the synthesis represent the enzymes: 1, ALAS; 1a, GTR; 1b, GSA-AT; 2, ALAD; 3, PBGD; 4, UROS; 5, UROD; 6, CPOX; 7, PPOX; 8, FeCH. The abbreviations of the enzyme names are explained in the text. The colors of the enzymatic steps stand for the origin of the genes, which are inferred from the phylogenetic trees (see Supplemental Figure 1 online). *Only incomplete information is available: Schizochytrium possesses ALAS, Perkinsus uses the C4 pathway as well as ALAD and UROD of the same origin as in primary heterotrophs. From Oxyrrhis marina, the sequence of the gene encoding PBGD shows a relation to the phototrophic PBGDs.

Figure 4.

Incorporation of Radiolabeled Amino Acids into Chlorophyll in C. velia and Cyanobacterium Synechocystis 6803. Cells were incubated with 350 μM ¹⁴C-Glu or ¹⁴C-Gly (specific activity ~50 mCi/mmol) for 2 h at 30°C and at 80 μmol of photons s⁻¹ m⁻². Chlorophyll was than extracted and converted into chlorin lacking both the phytol and methyl group at isocyclic E ring; thus, contains carbons from ALA precursors only. Prepared chlorin was separated by thin layer chromatography and exposed to an x-ray film. [See online article for color version of this figure.]