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

Abstract

AbstractProteins are heteropolymers composed of twenty standard amino acids. However, over 500 non-proteogenic amino acids exist in nature that can get misincorporated into proteins. Canavanine is an antimetabolite of L-arginine, with which it shares high chemical similarity. It can be utilized by bacteria such as Pseudomonas canavaninivorans in the legume rhizome as a sole source of carbon and nitrogen. However, canavanine is also incorporated in proteins of this bacterium as its arginyl-tRNA synthetase loads tRNAArg with both canavanine and arginine. The recently discovered canavanyl-tRNAArg deacetylase (CtdA) removes canavanine from misloaded tRNAArg and thereby prevents its incorporation in proteins. CtdA is the first enzyme known to edit tRNA mischarged with a non-proteinogenic amino acid. We have elucidated its crystal structure to 1.5 [A] resolution and studied its active site using site-directed mutagenesis. We found that CtdA is a small monomeric enzyme that presents a central, deep cavity that predictably constitutes the canavanine binding site and a positively charged surface area that likely coordinates the CCA-3 tRNA attachment sequence. The stand-alone, trans-editing CtdA is distantly related to the B3/B4 cis-editing domains of the large multi-subunit enzyme Phenylalanine tRNA synthetase (PheRS). Our comparative study reveals that CdtA and B3/B4 domains from bacterial and archeal/eukaryotic origin are three subclasses of a same conserved 3D-fold that differ in type-specific indels, which distinctly shape the substrate binding cleft of these proteins. We propose a unifying nomenclature of secondary structure elements for this 3D-fold. In CtdA, residues E191, Y104, N105 and E118 prove to be relevant for catalysis, of which N105 is conserved in bacterial B3/B4 domains. No other shared residues of catalytic relevance could be identified across enzymes of this class, so that a shared mechanism of catalysis appears unlikely in these editing enzymes.