Adaptation of the Romanomermis culicivorax CCA-Adding Enzyme to Miniaturized Armless tRNA Substrates

Abstract

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that—when inserted into the human enzyme—confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3′-end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

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

“…Concerning (t)RNA substrates, Anc CCA1 shows a comparably high specificity, as it accepts only conventional tRNA structures, but not bizarre substrates such as armless tRNAs recently identified in Enoplea ( supplementary fig. S4, Supplementary Material online; Hennig et al 2020 ). In addition, substrate RNAs artificially selected for modern CCA-adding enzymes ( Wende et al 2015 ) were also not accepted by the ancestral candidate.…”

“…While class 2 enzymes catalyze the CCA polymerization at high fidelity and efficiency, it is a mystery why these enzymes exhibit a rather weak affinity toward the tRNA substrate, so that in most cases, binding constants could not be determined ( Shi, Maizels, et al 1998 ; Tretbar et al 2011 ; Ernst et al 2015 ; Hennig et al 2020 ). In contrast, in class 1 enzymes, binding constants in a range of 30–700 nM are described ( Shi, Maizels, et al 1998 ; Okabe et al 2003 ; Cho et al 2005 , 2008 ).…”

“…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.…”