Unusual evolution of a catalytic core element in CCA-adding enzymes

Abstract

CCA-adding enzymes are polymerases existing in two distinct enzyme classes that both synthesize the C-C-A triplet at tRNA 3′-ends. Class II enzymes (found in bacteria and eukaryotes) carry a flexible loop in their catalytic core required for switching the specificity of the nucleotide binding pocket from CTP- to ATP-recognition. Despite this important function, the loop sequence varies strongly between individual class II CCA-adding enzymes. To investigate whether this loop operates as a discrete functional entity or whether it depends on the sequence context of the enzyme, we introduced reciprocal loop replacements in several enzymes. Surprisingly, many of these replacements are incompatible with enzymatic activity and inhibit ATP-incorporation. A phylogenetic analysis revealed the existence of conserved loop families. Loop replacements within families did not interfere with enzymatic activity, indicating that the loop function depends on a sequence context specific for individual enzyme families. Accordingly, modeling experiments suggest specific interactions of loop positions with important elements of the protein, forming a lever-like structure. Hence, although being part of the enzyme’s catalytic core, the loop region follows an extraordinary evolutionary path, independent of other highly conserved catalytic core elements, but depending on specific sequence features in the context of the individual enzymes.

Domain movements during CCA-addition: A new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases

“…To rule out that the reduced A-addition represents an effect specific to the tRNA Tyr substrate, the determination of MichaelisMenten kinetic parameters was conducted with another substrate, yeast tRNA Phe , one of the most commonly used substrates in CCA adding reactions. 16,17,21 To analyze the efficiency of the terminal A-addition. While the observed K M values of 0.09 mM and 0.11 mM did not differ significantly between the wt enzyme and the D139A variant (p D 0.8), the latter variant showed a strongly reduced k cat value of 0.008 s ¡1 , compared to 0.119 s…”

“…6,8,12,30 In addition to this sequence signature, a highly flexible loop element, located between motifs A and B, is involved in switching the enzyme's specificity from C-towards A-addition during synthesis. 16,17 For four of the five signature motifs, a specific function could be identified, while motif C represents one of the less characterized catalytic core elements. [31][32][33][34][35] Molecular modeling data of the human CCA-adding enzyme (HsaCCA) as well as the crystal structure of a closely related A-adding enzyme form A. aeolicus excluded a direct interaction with tRNA or nucleotide substrates (Fig.…”

“…[31][32][33][34][35] Molecular modeling data of the human CCA-adding enzyme (HsaCCA) as well as the crystal structure of a closely related A-adding enzyme form A. aeolicus excluded a direct interaction with tRNA or nucleotide substrates (Fig. 1B) 10, 16 The first evidence that motif C contributes to an efficient CCA-addition came from the analysis of a temperature-sensitive mutation in the yeast CCA-adding enzyme. Phe -CC to 1 mM concentration, the fluorescence spectra of both enzyme forms show identical signal quenching (according to student's t-test, none of the values show a significant difference between wt and D139A variant).…”

“…16,17 Figure 2. Biochemical analysis of conserved amino acids in motif C of the human CCA-adding enzyme.…”

“…This loop seems to interact with the amino acid template of motif D, and biochemical as well as molecular modeling data support a function as a lever in order to adjust the templating amino acids for CTP or ATP specificity. 16 Interestingly, this loop is missing in the above mentioned CCadding enzymes, explaining the restricted activity of these nucleotidyltransferases. 17 While the connecting motif C shows a rather low level of sequence conservation with only three predominant positions (Dx (3,4) Gx (9) R), mutations therein can cause a temperaturesensitive phenotype, indicating an important role in catalysis.…”