Unusual Occurrence of Two Bona-Fide CCA-Adding Enzymes in Dictyostelium discoideum

Abstract

Dictyostelium discoideum, the model organism for the evolutionary supergroup of Amoebozoa, is a social amoeba that, upon starvation, undergoes transition from a unicellular to a multicellular organism. In its genome, we identified two genes encoding for tRNA nucleotidyltransferases. Such pairs of tRNA nucleotidyltransferases usually represent collaborating partial activities catalyzing CC- and A-addition to the tRNA 3′-end, respectively. In D. discoideum, however, both enzymes exhibit identical activities, representing bona-fide CCA-adding enzymes. Detailed characterization of the corresponding activities revealed that both enzymes seem to be essential and are regulated inversely during different developmental stages of D. discoideum. Intriguingly, this is the first description of two functionally equivalent CCA-adding enzymes using the same set of tRNAs and showing a similar distribution within the cell. This situation seems to be a common feature in Dictyostelia, as other members of this phylum carry similar pairs of tRNA nucleotidyltransferase genes in their genome.

CCA-addition in the cold: Structural characterization of the psychrophilic CCA-adding enzyme from the permafrost bacterium Planococcus halocryophilus

“…S5 ). For Gst CCA, an interaction with a tRNA substrate was clearly visible, resulting in a Kd value of approximately 1.3 µM, comparable to that of other class II CCA-adding enzymes [24] , [25] . In contrast, no gel shift was visible for Pha CCA, indicating a reduced tRNA interaction that does not allow a calculation of a binding constant.…”

“…Electrophoretic mobility shift assays were performed as recently described with minor deviations [24] , [25] . 0.3 pmol α– 32 P–ATP-labeled yeast tRNA Phe lacking the CCA-terminus was heated for 2 min at 90 °C, cooled down to room temperature and incubated with 0 to 5 µM enzyme in 50 mM glycine/NaOH (pH 8.5), 10 mM MgCl 2 and 2 mM DTT for 10 min at temperatures where the enzymes show significant activity (4 °C and 30 °C for Pha CCA and 37 °C for Gst CCA) [22] .…”

Substrate Affinity Versus Catalytic Efficiency: Ancestral Sequence Reconstruction of tRNA Nucleotidyltransferases Solves an Enzyme Puzzle

“…To investigate the molecular reason for these obviously conflicting features of catalytic efficiency versus low substrate affinity, we set out to compare class 2 enzymes that differ in these parameters. However, only enzymes adapted to bizarre armless tRNA structures ( Hennig et al 2020 ) or unconventional enzymes with a series of Q/N insertions of unknown function exhibit a detectable affinity toward tRNA substrates ( Erber et al 2020 ). Hence, we used the approach of ASR to generate a conventional candidate enzyme with ancestral features that differ from modern enzymes in terms of substrate binding, while it is not adapted to bizarre tRNA substrates.…”

Changes of the tRNA Modification Pattern during the Development of Dictyostelium discoideum

“…Finally, the fruiting body contains a sorus of spores on top of a stalk which germinate following dispersal, renewing the cycle (24 h after starvation). The figure ist modified from [46].…”