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.

Figure 1.

Conservational analysis of the region surrounding the flexible loop in biochemically well-characterized CCA-adding enzymes. Using the Jalview software, the conservation levels of individual amino acid positions are indicated by colors [from dark red (high) to light orange (low)]. The flexible loop region shows almost no conservation, while motifs A and B carry highly invariant positions. A similar level of conservation was found for the catalytically active carboxylates (labeled by red asterisks) and the basic/acidic motif BxA (indicated by red dots) that is probably involved in primer positioning. The black bars indicate the two highly conserved signatures (upstream of the loop: BxA; downstream: RRD in motif B) as selected fusion positions for the reciprocal exchanges of the loop regions.

Figure 2.

Loop exchanges between CCA-adding enzymes of H. sapiens, E. coli and B. stearothermophilus. (A) Arrows indicate the loop transplantations leading to chimeras HEH, EHE and EBE. Fusion positions were selected as described in the text. Characters labeled in red display conserved positions of each loop sequence. (B) (Upper panel) Nucleotide incorporation into a radioactively labeled tRNA substrate catalyzed by the chimeric enzymes. While wt enzymes of E. coli and H. sapiens showed a complete CCA-addition leading to a corresponding shift in the migration position of the products, the chimeras incorporated only two residues, but failed to add the terminal adenosine. (Lower panel) Relative k_cat values for ATP-incorporation of investigated enzymes. The values of the wt enzymes were set to 100%. The chimeras showed relative values close to 0%, demonstrating a dramatic reduction of the A-adding activity, while the CTP-incorporation was not affected (data not shown).

Figure 3.

Data set cleaning procedure for the identification of CCA-adding enzyme sequences. The starting data set of class II nucleotidyltransferases was retrieved by a genomic BLAST analysis of the NCBI database, using sequences of experimentally verified CCA-adding enzymes. According to the identity motifs of the individual types of class II nucleotidyltransferases, this original data set was purified from other entries (CC- and A-adding enzymes, poly(A) polymerases) and finally contained 339 sequences that carried all identity elements of genuine CCA-adding enzymes. This purified data set was used for further analysis.

Figure 4.

Families of individual loop sequences. A phylogenetic tree based on rRNA sequences (41,42) was used to illustrate the loop consensus sequences identified for each of the presented genera. While some positions are found in several families, no amino acid residue is present at a certain position in all families, indicating that the loops do not contain absolutely conserved positions. The figures behind the sequence logos indicate the number of sequences of each genus that was used for this analysis. For the sequence alignment, Clustal W and Weblogo software was used. The analysis was restricted to the flexible loop region flanked by the conserved elements BxA (basic/acidic motif, N-terminal of the loop) and DxxRRD (motif B, C-terminal of the loop).

Figure 5.

Loop replacements within sequence families. (A) Enzymes of E. coli/W. glossinidia and H. sapiens/D. melanogaster were chosen as representatives for γ-Proteobacteria and Vertebrata/Arthropoda, leading to loop chimeras EWE and HDH, respectively. Fusion positions were selected as in Figure 2. Conserved positions in the individual loop sequences are indicated in red, introduced point mutations S69A, Y71A (both in E. coli), T101A and R105A (both in H. sapiens) are labeled by asterisks. (B) Nucleotide addition activity of chimeras and enzymes carrying point mutations. Both EWE and HDH chimeras show a full CCA-adding activity, indicating compatible loop sequences of the parental wt enzymes that belong to the same sequence families. The point mutations at highly conserved positions (Y71A, R105A) dramatically interfere with ATP-addition, while CTP-incorporation remains unaffected. Correspondingly, alanine replacements at nonconserved positions do not influence the overall activity of the enzymes. (C) Comparative analysis of k_cat values of the mutant enzymes. While the point mutations demonstrate that conserved loop positions are essential for A-addition, the loop exchanges within sequence families affect the A-incorporation only slightly.

Figure 6.

Molecular modeling of loop interactions. (A) Representation of the crystal structure of the human CCA-adding enzyme. The position of the flexible loop is indicated by the dashed blue line. The presentation is based on the pdb deposit number 1OU5 (7). (B) In the simulation, an arginine in the flexible loop (blue) forms a salt bridge to a glutamate residue of the nucleotide binding pocket (red), changing the enzyme from an open (upper panel) to a closed conformation (lower panel). (C) Time course of the simulation. After an initial phase of 1 ns (gray), the salt bridge with a distance of about 4 Å between R105 and E164 is formed, leading to a closed conformation of the enzyme (black filled arrow). Short interruptions of this interaction lead to the open conformation with a distance of 8–12 Å between these residues (open arrow), indicating the dynamic nature of this interaction.