The crystal structure of an enzymesubstrate complex with histidyl-tRNA synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme-product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryltRNA synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (k cat ͞K m ) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl 2 reveal the existence of two metal binding sites between ␤-and ␥-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases.Before their ligation to the 3Ј ends of tRNAs, amino acids must be activated by condensation with ATP. This aminoacyl adenylylation reaction is catalyzed by the aminoacyl-tRNA synthetases (aaRS), which are divided into two classes of 10 members each that are based on two different catalytic folds (1, 2). Each class possesses diagnostic sequence motifs whose conserved residues are necessary for ATP binding (3, 4). Based on the crystallographic analyses of complexes of aaRS with ATP or their respective adenylates, ATP recognition is largely mediated through residues that are invariant among class members, whereas amino acid recognition occurs by using residues that are unique to the evolutionary family (5-14). For most systems, it has not yet been possible to obtain complexes containing both amino acid and unreacted ATP (or suitable analog), so significant mechanistic issues remain to be addressed, including the precise roles of specific catalytic residues, bound metal ions, and mobile loop elements. To provide further insight into these issues, we have characterized the structures of two different enzyme-substrate complexes of histidyl-tRNA synthetase (HisRS) from Escherichia coli, one with the histidyl-adenylate and the other with ATP and histidinol. In addition, data were collected from crystals into which MnCl 2 had been diffused to provide information about divalent metal binding sites. Analysis of these complexes provides evidence for both general features of the catalytic mechanism of class II enzymes and features that are specific to the histidine system.

Identity elements in tRNAs and the intracellular balance of tRNAs allow accurate selection of tRNAs by aminoacyl-tRNA synthetases. The histidyl-tRNA from Escherichia coli is distinguished by a unique G-1.C73 base pair that upon exchange with other nucleotides leads to a marked decrease in the rate of aminoacylation in vitro. G-1.C73 is also a major identity element for histidine acceptance, such that the substitution of C73 brings about mischarging by glycyl-, glutaminyl-, and leucyl-tRNA synthetases. These identity conversions mediated by the G-1.C73 base pair were exploited to isolate secondary site revertants in the histidyl-tRNA synthetase from E. coli which restore histidine identity to a histidyl-tRNA suppressor carrying U73. The revertant substitutions confer a 3-4 fold reduction in the Michaelis constant for tRNAs carrying the amber-suppressing anticodon and map to the C-terminal domain of HisRS and its interface with the catalytic core. These findings demonstrate that the histidine tRNA anticodon plays a significant role in tRNA selection in vivo and that the C-terminal domain of HisRS is in large part responsible for recognizing this trinucleotide. The kinetic parameters determined also show a small degree of anticooperativity (delta delta G = -1.24 kcal/mol) between recognition of the discriminator base and the anticodon, suggesting that the two helical domains of the tRNA are not recognized independently. We propose that these effects substantially account for the ability of small changes in tRNA binding far removed from the site of a major determinant to bring about a complete conversion of tRNA identity.

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Primordial aminoacyl-tRNA synthetases (aaRSs) based on the Rossman nucleotide binding fold of class I enzymes or the seven-stranded antiparallel beta-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 (Ncat HisRS) truncated immediately following motif 3 catalyzes both the specific aminoacylation of tRNA and pyrophosphate exchange, albeit less efficiently than the full-length enzyme. Ncat 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 Ncat 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 (Cter HisRS) was inactive for both reactions and did not enhance the activity of Ncat 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.