DESIGN OF AN ACTIVE FRAGMENT OF A CLASS-II AMINOACYL-TRANSFER-RNA SYNTHETASE AND ITS SIGNIFICANCE FOR SYNTHETASE EVOLUTION

Primordial aminoacyl-tRNA synthetases (aaRSs) based on the Rossman nuc leotide binding fold of class I enzymes or the seven-stranded antipara llel beta-sheet fold of class II enzymes have been proposed to predate the contemporary aaRS. As part of an inquiry into class II aaRS evolu tion, the individual domains of the homodimeric Escherichia coli histi dyl-tRNA synthetase (HisRS) were separately expressed and purified to determine their individual contributions to catalysis. A 320-residue f ragment (N-cat HisRS) truncated immediately following motif 3 catalyze s both the specific aminoacylation of tRNA and pyrophosphate exchange, albeit less efficiently than the full-length enzyme, N-cat HisRS show ed no mischarging of noncognate tRNAs but exhibited reduced selectivit y for the C73 discriminator base, a principal aminoacylation determina nt for histidine tRNAs. Size exclusion chromatography showed that N-ca t HisRS is monomeric, indicating that the C-terminal domain is essenti al for maintaining the dimeric structure of the enzyme. The stably fol ded C-terminal domain (C-ter HisRS) was inactive for both reactions an d did not enhance the activity of N-cat HisRS when added in trans. The fusion of one or more accessory domains to a primordial catalytic dom ain may therefore have been a critical evolutionary step by which amin oacyl-tRNA synthetases acquired increased catalytic efficiency and sub strate specificity.