Indel-driven evolution of the canavanine tRNA-editing deacetylase enzyme CtdA

Proteins are heteropolymers composed of twenty standard amino acids, but over 500 non-proteogenic amino acids exist in nature that can be misincorporated into proteins. Canavanine is an antimetabolite of the chemically similar L-arginine. It can be utilized by bacteria such as <i>Pseudomonas canavaninivorans</i> in the legume rhizome as a sole source of carbon and nitrogen. However, canavanine misincorporates in proteins of this bacterium as its arginyl-tRNA synthetase loads tRNA^Arg with both canavanine and arginine. Canavanyl-tRNA^Arg deacetylase (CtdA) removes canavanine from misloaded tRNA^Arg, preventing its protein toxicity, being the first enzyme known to edit tRNA mischarged with a non-proteinogenic amino acid. We have elucidated CtdA's crystal structure and studied its active site using site-directed mutagenesis. We found that CtdA is a small monomeric enzyme with a central, deep cavity that predictably is the canavanine binding site and a positively charged surface area that likely coordinates the CCA-3' tRNA attachment sequence. CtdA is distantly related to the B3/B4 <i>cis</i>-editing domains of the multi-subunit enzyme Phenylalanine-tRNA-Synthetase (PheRS). CdtA and B3/B4 domains from bacterial and archaeal/eukaryotic origin are three subclasses of a conserved 3D-fold that differ in type-specific indels, which shape the substrate binding site. We propose a class-unifying nomenclature of secondary structure for this fold. In CtdA, residues Y104, N105, E118 and E191 are relevant for catalysis, of which N105 is conserved in bacterial B3/B4 domains. Residue N105 is in proximity of the canavanyl-ribose junction and might coordinate the nucleophilic water molecule that attacks the substrate, possibly sharing a mechanistic role in CtdA and bacterial B3/B4 editing enzymes.