Catalase and Ascorbate Peroxidase in Euglenozoan Protists

Abstract

In this work, we studied the biochemical properties and evolutionary histories of catalase (CAT) and ascorbate peroxidase (APX), two central enzymes of reactive oxygen species detoxification, across the highly diverse clade Eugenozoa. This clade encompasses free-living phototrophic and heterotrophic flagellates, as well as obligate parasites of insects, vertebrates, and plants. We present evidence of several independent acquisitions of CAT by horizontal gene transfers and evolutionary novelties associated with the APX presence. We posit that Euglenozoa recruit these detoxifying enzymes for specific molecular tasks, such as photosynthesis in euglenids and membrane-bound peroxidase activity in kinetoplastids and some diplonemids.

Keywords: Euglenozoa; ascorbate peroxidase; catalase; enzymatic activity; phylogeny.

Figure 1

Maximum-likelihood phylogeny of heme peroxidases possessing APX domains in Euglenozoa. Taxa representing euglenozoan sequences are marked by symbols according to the graphical legend. Full tree in Newick format can be found in File S1.

Figure 2

Maximum-likelihood phylogeny of catalases in Euglenozoa. (A) Unrooted tree showing ancestral origin of diplonemid CAT branching within eukaryotes and three horizontal gene transfer events in Blastocrithidia/“jaculum”, Leishmaninae and D. papillatum. Full tree in Newick format can be found in File S2. (B) Subtree showing the ancestral diplonemid CAT. (C) Subtree showing the D. papillatum CAT related to alpha-proteobacteria. UFBoot support values are shown when ≥ 80.

Figure 3

Biochemical and transcriptomic analysis. Comparison of (A) APX and (C) CAT activities, (B) APX and (D) CAT expression levels, and (E) oxygen uptake in Diplonema papillatum cultivated in nutrient-rich (R) and nutrient-poor (P) medium, Rhynchopus humris, Blastocrithidia sp. P57, Leptomonas seymouri cultivated at 14 °C (14), 23 °C (23) and 34 °C (34), Crithidia thermophila cultivated at 14 °C (14), 23 °C (23) and 34 °C (34), Novymonas esmeraldas, Trypanosoma brucei, Euglena gracilis cultivated in light (L) and dark (D), and Euglena longa. Species names in grey denote organisms, in which corresponding enzyme was not identified. Activity U is defined as the amount of the enzyme which catalyzes the conversion of 1 μmol of ascorbate (APX) or H₂O₂ (CAT) per 1 min. Each experiment was performed in two biological replicates. Statistical significance of differences between organisms was evaluated by an unpaired t-test. * statistically significant (p < 0.05). Note that respiration value in light-grown E. gracilis is masked by photosynthetic oxygen consumption (grey bar).

Figure 4

Schematic alignment of APX sequences from selected taxa. APXs from organisms studied previously are boxed in grey. Important amino acid residues are highlighted by different colors explained in the graphical legend.