Phylogenomic analysis of integral diiron membrane histidine motif-containing enzymes in ciliates provides insights into their function and evolutionary relationships

Abstract

The Integral Membrane Histidine Motif-containing Enzymes (IMHME) are a class of binuclear non-heme iron proteins widely distributed among prokaryotes and eukaryotes. They are characterized by a conserved tripartite motif consisting of eight to ten histidine residues. Their known function is the activation of the dioxygen moiety to serve as efficient catalysts for reactions of hydroxylation, desaturation or reduction. To date most studies on IMHME were carried out in metazoan, phototrophic or parasitic organisms, whereas genome-wide analysis in heterotrophic free living protozoa, such as the Ciliophora phylum, has not been undertaken. In the seven fully sequenced genomes available we retrieved 118 putative sequences of the IMHME type, albeit with large differences in number among the ciliates: 11 sequences in Euplotes octocarinatus, 7 in Ichthyophthirius multifiliis, 13 in Oxytricha trifallax, 18 in Stylonychia lemnae, 25 in Tetrahymena thermophila, 31 in Paramecium tetraurelia and 13 in Pseudocohnilembus persalinus. The pool of putative sequences was classified in 16 orthologous groups from which 11 were related to fatty acid desaturase (FAD) and 5 to the fatty acid hydroxylase (FAH) superfamilies. Noteworthy, a large diversity on the number and type of FAD / FAH proteins were found among the ciliates, a feature that, in principle, may be attributed to peculiarities of the evolutionary process, such as gene expansion and reduction, but also to horizontal gene transfer, as we demonstrate in this work. We identified twelve putative enzymatic activities, from which four were newly assigned activities: sphingolipid Δ4-desaturase, ω3/Δ15 fatty acid desaturase, a large group of alkane 1-monooxygenases, and acylamide-delta-3(E)-desaturase, although unequivocal allocation would require additional experiments. We also combined the phylogenetics analysis with lipids analysis, thereby allowing the detection of two enzymatic activities not previously reported: a C-5 sterol desaturase in P. tetraurelia and a delta-9 fatty acid desaturase in Cohnilembus reniformis. The analysis revealed a significant lower number of FAD's sequences in the spirotrichea ciliates than in the oligohymenophorea, emphasizing the importance of fatty acids trophic transfer among aquatic organisms as a source of variation in metabolic activity, individual and population growth rates, and reproduction.

Genome‐wide Transcriptional Analysis of Tetrahymena thermophila Response to Exogenous Cholesterol

“…T. thermophila 's genome encodes 16 putative genes for oxygenases belonging to the long spacing subfamily of FAHD (section 3.5.2) (Cid et al. ). Five of them were differentially expressed (Table ) in the presence of cholesterol: (1) TTHERM_00334320 was downregulated (−5.056 fold) and showed similarity to sphingolipid desaturases; (2) TTHERM_00052620 was induced 2.490‐fold and is most probably a stearoyl‐CoA desaturase, sharing 30% identity with Saccharomyces cerevisiae OLE1; (3) TTHERM_00339850 was induced 5.137‐fold and shares 58% identity with a paralog (TTHERM_00138530) recently characterized as Δ6 linoleoyl desaturase (Dahmen et al.…”

“…In previous studies, these two sequences were grouped together with other two paralogous genes that are not differentially expressed in our dataset (TTHERM_01133950 and TTHERM_00331050), and with fungal acylamide delta‐3 desaturases (Cid et al. ). These enzymes, only characterized in fungi, introduce a double bond at the C‐3 position of the hydroxyl‐fatty acyl moiety of sphingolipids (Cid et al.…”

“…These enzymes, only characterized in fungi, introduce a double bond at the C‐3 position of the hydroxyl‐fatty acyl moiety of sphingolipids (Cid et al. ). To date, no delta‐3 unsaturated compound, neither as the free fatty acid nor in the fatty acyl moiety of sphingolipids or triglycerides, has been found in Tetrahymena (Cid et al.…”

“…With this in mind, we searched for putative oxygenases among the DEGs in our RNA-seq dataset. T. thermophila's genome encodes 16 putative genes for oxygenases belonging to the long spacing subfamily of FAHD (section 3.5.2) (Cid et al 2017). Five of them were differentially expressed (Table S4) in the presence of cholesterol: (1) TTHERM_00334320 was downregulated (À5.056 fold) and showed similarity to sphingolipid desaturases; (2) TTHERM_00052620 was induced 2.490-fold and is most probably a stearoyl-CoA desaturase, sharing 30% identity with Saccharomyces cerevisiae OLE1;…”

“…(3) TTHERM_00339850 was induced 5.137-fold and shares 58% identity with a paralog (TTHERM_00138530) recently characterized as D6 linoleoyl desaturase (Dahmen et al 2013); and (4) the last two genes, TTHERM_00085010 and TTHERM_00129290 have 56% identity, they have no clear homology to described enzymes and are the second (75.412-fold) and third (70.078-fold) most upregulated genes in our dataset, respectively. In previous studies, these two sequences were grouped together with other two paralogous genes that are not differentially expressed in our dataset (TTHERM_01133950 and TTHERM_00331050), and with fungal acylamide delta-3 desaturases (Cid et al 2017). These enzymes, only characterized in fungi, introduce a double bond at the C-3 position of the hydroxyl-fatty acyl moiety of sphingolipids (Cid et al 2017).…”