Advvances in Protein Chemistry and Structural Biology

Advances in Protein Chemistry and Structural Biology, 2012

Microbiology and Molecular Biology Reviews, 2003

2009

Acta biochimica Polonica, 2000

Aminoacyl-tRNA synthetases (AARS) are essential proteins found in all living organisms. They form a diverse group of enzymes that ensure the fidelity of transfer of genetic information from the DNA into the protein. AARS catalyse the attachment of amino acids to transfer RNAs and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid. Here we summarise the effects of recent studies on this interesting family of multifunctional enzymes.

Trends in Biochemical Sciences, 1997

The Journal of biological chemistry, 1991

1

Microbiology and Molecular Biology Reviews, 2000

Biochemistry, 1997

Primordial aminoacyl-tRNA synthetases (aaRSs) based on the Rossman nucleotide binding fold of class I enzymes or the seven-stranded antiparallel -sheet fold of class II enzymes have been proposed to predate the contemporary aaRS. As part of an inquiry into class II aaRS evolution, the individual domains of the homodimeric Escherichia coli histidyl-tRNA synthetase (HisRS) were separately expressed and purified to determine their individual contributions to catalysis. A 320-residue fragment (N cat HisRS) truncated immediately following motif 3 catalyzes both the specific aminoacylation of tRNA and pyrophosphate exchange, albeit less efficiently than the full-length enzyme. N cat HisRS showed no mischarging of noncognate tRNAs but exhibited reduced selectivity for the C73 discriminator base, a principal aminoacylation determinant for histidine tRNAs. Size exclusion chromatography showed that N cat HisRS is monomeric, indicating that the C-terminal domain is essential for maintaining the dimeric structure of the enzyme. The stably folded C-terminal domain (C ter HisRS) was inactive for both reactions and 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 domain may therefore have been a critical evolutionary step by which aminoacyl-tRNA synthetases acquired increased catalytic efficiency and substrate specificity. X Abstract published in AdVance ACS Abstracts, March 15, 1997. 1 Abbreviations: aaRS, aminoacyl-tRNA synthetase; HPLC, highpressure liquid chromatography; IPTG, isopropyl 1-thio--D-galactopyranoside; HEPES, N-(2-hydroxyethyl)piperazine-N ′-2-ethanesulfonic acid; Ni-NTA, nickel nitrilotriacetic acid; NOESY, nuclear Overhauser enhancement spectroscopy; PCR, polymerase chain reaction. Following the standard convention, aminoacyl-tRNA synthetases are referred to by the three-letter code for their amino acid substrate, followed by the suffix RS.

J Mol Biol, 2000

Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNA Tyr aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA Tyr. Previous investigations indicate that the class I conserved KMSKS motif is involved in only the ®rst step of the reaction (i.e. tyrosine activation). Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step. In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction. These results imply that stabilization of the transition state for the ®rst step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 3 H end of tRNA. We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP.