Figure S5: Comparison of metabolic pathways between Danio rerio (zebrafish ) and the three ciliates Tetrahymena thermophila, Paramecium tetraurelia and Ichthyophthirius multifiliis (Ich).

Mappings were done on KEGG metabolic pathway maps using the color pathways tools (http://www.genome.jp/kegg/tool/color_pathway.html).  Four digit EC numbers from all four species were painted on the same map and color-coded to distinguish the following categories:

Grey = enzyme present in all four species

Grey + Blue foreground = enzyme present in D. rerio and at least one other ciliate

Cyan = enzyme present in D. rerio but absent in all three ciliates

Yellow = enzyme present in all three ciliates but absent in D. rerio

Yellow + Green foreground = enzyme absent in D. rerio and Ich but present in one other ciliate

Yellow + Red foreground = enzyme absent in D. rerio but present in Ich and one other ciliate

Clicking on the hyperlinked KEGG pathway numbers below allows viewing of the color-coded enzyme mapping.  The notes below are a brief summary of the status of each pathway in the four species.

Carbohydrate Metabolism

00010  Glycolysis/Gluconeogenesis: Glycolysis the same in all four species and all of them can synthesize all glycolytic metabolites by gluconeogenesis (i.e. starting with oxaloacetic acid and ending with glucose-6-phosphate)

00020  Citric Acid Cycle cycle (TCA cycle): Same in all four species; pyruvate dehydrogenase present in all. Interestingly, all three ciliates (but not D. rerio) have isocitrate lyase and malate synthase (glyoxoalate cycle – see below). These enzymes provide a bypass in the TCA cycle allowing conversion of isocitrate directly to succinate and malate.  This avoids the CO₂-producing steps allowing more efficient assimilation of carbon from fatty acids via acetyl-CoA. 

00630 Glyoxalate and dicarboxylate metabolism: All three ciliates have the glyoxalate cycle which is absent in D. rerio.

00030  Pentose phosphate pathway: All three ciliates lack the first two enzymes of this pathway that generates NADPH. What other pathways exists in ciliates for maintaining their cytosolic NADPH levels in the absence of these pentose phosphate pathway enzymes is not clear.

00500  Starch and sucrose metabolism: All three ciliates are capable of synthesizing starch/amylopectin but not glycogen. D. rerio can make glycogen but not starch/amyopectin.

00520  Amino sugar and nucleotide sugar metabolism: All four species can synthesize UDP-glucose, UDP-galactose and uridine diphosphate N-acetylglucosamine. D. rerio has a more comprehensive set of enzymes and can make GDP-fucose & neuraminic acid.

00620  Pyruvate metabolism: Same in all four species.

00640  Propanoate metabolism: Major differences between D. rerio and ciliates. D. rerio metabolizes propionyl-CoA via the methylmalony-CoA pathway. Ciliates metabolize propionyl-CoA via the methyl-citric acid cycle.  Enzymes of this pathway have been studied as therapeutic targets (e.g. in Mycobacterium tuberculosis).

00562  Inositol phosphate metabolism: Essentially the same in all four species with the exception that the Ich gene for CDP-diacylglycerol-inositol 3-phosphatidyltransferase (2.7.8.11), could not be found, possibly because it is located in a gap in the assembly or due to an incorrect gene model. This enzyme is required for making phosphatidyl-inositol-monophosphate from which all other phosphorylated versions are made (see below in glycerophospholipid metabolism for this enzyme mapping). 

Energy Metabolism

00190  Oxidative phosphorylation and F₁F₀-ATP synthase: Pathway present in all four species.  Ciliates have an unusual F₁F₀-ATP synthase subunit composition (see text).  Therefore this enzyme is a potential therapeutic target.

00910  Nitrogen metabolism: No major differences between species

00920  Sulfur metabolism: Only D. rerio and Ich are capable of synthesizing phosphoadenylyl sulfate.  Only Ich has the cysteine synthase enzyme which can use H₂S as a sulfur donor for making cysteine.

Lipid Metabolism

00061  Fatty acid biosynthesis: Major differences between D. rerio and ciliates. All three ciliates lack both the Type I and II pathways for fatty acid biosynthesis.  D. rerio has the multifunctional Type I polypeptide for fatty acid synthesis.  Paramecium has a probable polyketide synthase that is a similar multifunctional enzyme to Type I FAS polypeptide (explaining why the coloring is grey + blue foreground for this enzyme as KEGG does not distinguish between the FAS I & PKS multifuncaitonal enzymes)

00062  Fatty acid elongation in mitochondria: All four species are capable of elongating fatty acids.  00071  Fatty acid metabolism: All four species have all the enzymes required for fatty acid breakdown via beta-oxidation.

00072  Synthesis and degradation of ketone bodies: Same in all four species.

00100  Steroid biosynthesis: Only D. rerio can convert the terpenoid isoprene into cholesterol and other steroid derivatives.  Ciliates cannot synthesize cholesterol or other steroids but can modify them.  For example ciliates can esterify cholestrol to cholestyl-esters.

00561  Glycerolipid metabolism: D. rerio, Ich and Tetrahymena have similar pathways for mono-, di- and tri-acylglycerol metabolism.

00564  Glycerophospholipid metabolism: D. rerio can make all phospholipids; Ich can make phosphatidyl-choline, phosphatidyl-ethanolamine and phosphatidyl-serine, but seems to be missing the enzyme required to make phosphatidyl-inositol (see above in inositol metabolism).

00600  Sphingolipid metabolism: No major difference between the four species.

01040  Biosynthesis of unsaturated fatty acids: All species are capable of fatty acid desaturation.

Nucleotide Metabolism

00230  Purine metabolism: Unlike D. rerio, ciliates are not capable of synthesizing purines, depending instead on salvage reactions. There are also interesting differences among ciliates.  Cilliates cannot convert IMP to GMP and so have to scavenge external guanine or guanosine.  Only Ich is incapable of making AMP either from adenine (Paramecium can do this) or adenosine (Paramecium and Tetrahymena can do this).

00240  Pyrimidine metabolism: Unlike D. rerio, ciliates are incapable of synthesizing pyrimidines and depend on pyrimidine salvage for survival. In particular, uracil is made from cytosine in ciliates using cytosine deaminase, a possible therapeutic target.

Amino Acid Metabolism

00250  Alanine, aspartate and glutamate metabolism: No major difference between species.     

00260  Glycine, serine and threonine metabolism: No major difference between species.

00270  Cysteine and methionine metabolism: No major difference between species.

00280  Valine, leucine and isoleucine degradation: No major difference between species.

00290  Valine, leucine and isoleucine biosynthesis: No major difference between species.

00300  Lysine biosynthesis: None of the four species can synthesize lysine.

00310  Lysine degradation: D. rerio, Ich and Tetrahymena have similar sets of  enzymes for lysine degradation, which appear to be lacking in Paramecium.

00330  Arginine and proline metabolism: D. rerio carries out the urea cycle, but this pathway is missing in all ciliates.  D. rerio, Ich and Tetrahymena, but not Paramecium, are capable of synthesizing proline from ornithine. Polyamine pathway: D. rerio, Ich and Tetrahymena can convert ornithine to putrescine, then to spermidine and then spermine.  Paramecium can make putrescine but not spermidine and spermine.

00340  Histidine metabolism: When compared to D. rerio, ciliates have limited histidine metabolism but possess the auromatic amino acid decarbosylase (AAD) enzyme that is capable of synthesizing histamine, although the biological relevance of this is not clear.

00350  Tyrosine metabolism: D. rerio and ciliates can break down tyrosine to acetoacetate (and with help of the AAD enzyme can decarboxylate tyrosine to tyramine & can potentially convert L-DOPA to dopamine, although they cannot synthesize L-DOPA).

00360  Phenylalanine metabolism: No major difference between species.

00380  Tryptophan metabolism: No major difference between species. Interestingly ciliates are capable of converting tryptophan to serotonine and tryptamine using the AAD enzyme (see above in histidine and tyrosine metabolism).

00400  Phenylalanine, tyrosine and tryptophan biosynthesis: All three ciliates have the AROM pentafunctional polypeptide which is part of the shikimic acid pathway for chorismate biosynthesis.  D. rerio does not have this enzymes or the pathway, making this pathway a potential drug target.

Metabolism of Other Amino Acids

00450  Selenocysteine metabolism: D. rerio and the three ciliates differ in how they make Se-Cys.  D. rerio can make Se-Cys from H₂Se and acetyl-serine but ciliates can make Se-Cys from acetyl-serine only.

00480  Glutathione metabolism: All four species have the same pathway for glutathione synthesis and utilization via the oxidation/reduction cycle. Interestingly, all three ciliates seem to be capable of synthesizing trypanothione and utilizing it in oxidation/reduction cycle.

Metabolism of Cofactors and Vitamins

00670  One carbon pool by folate: No major difference between species.

00860  Porphyrin and chlorophyll metabolism: All four species use the animal/fungal type C4 pathway for heme biosynthesis.

00785 Lipoic acid metabolism: Ich and other ciliates have only the salvage pathway but not the biosynthetic pathway for lipoic acid.  D. rerio has both.

Others

00900  Terpenoid backbone biosynthesis: No difference.  All four species make the terpenoid backbone using the mevalonate pathway.

00563  Glycosylphosphatidylinositol(GPI)-anchor biosynthesis: All four species are capable of GPI anchor biosynthesis. NOTE: many enzymes of this pathway have only three digit EC numbers.  So for Ich mapping these were left out as only four digit EC numbers were used. However, ortholog mapping suggests that most enzymes present in Tetrahymena are also present in ich.