Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor.
Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNA(Gln) is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNA(Gln), and an amidotransferase converts Glu-tRNA(Gln) to Gln-tRNA(Gln). The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNA(Gln). It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Li-Tao
LT
Guo
Sunna
S
Helgadóttir
Dieter
D
Söll
Jiqiang
J
Ling
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology; D005975:Glutamate-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology; D008969:Molecular Sequence Data; D015202:Protein Engineering; D012351:RNA, Transfer, Gln / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D046249:Transfer RNA Aminoacylation
D012351:RNA, Transfer, Gln; D018698:Glutamic Acid; D000604:Amino Acyl-tRNA Synthetases; D005975:Glutamate-tRNA Ligase; C020659:glutaminyl-tRNA synthetase