Valentina N. Ankilova

Phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 Purification and properties of the crystallizing enzyme

Phenylalanyl‐tRNA synthetase from Thermus thermophilus HB8 was isolated, characterized and crystallized. The enzyme is a tetramer of α2β2‐type structure, its molecular mass being 264 kDa. Molecular masses of the enzyme subunits are 40 (α) and 92 (β) kDa. The optimal temperature conditions of the tRNAPhe aminoacylation, catalyzed by this enzyme, are close to 80°C. K M values for tRNAPhe from E.coli, for tRNAPhe from T. thermophilus HB8, for phenylalanine and ATP, as well as their temperature dependencies were determined. The enzyme crystals were grown by the hanging drop technique at 4°C in the presence of ammonium sulfate.

Structure at 2.6 Å resolution of phenylalanyl-tRNA synthetase complexed with phenylalanyl-adenylate in the presence of manganese

The crystal structure of phenylalanyl-tRNA synthetase (PheRS) from Thermus thermophilus, a class II aminoacyl-tRNA synthetase, complexed with phenylalanyl-adenylate (Phe-AMP) was determined at 2.6 A resolution. Crystals of native PheRS were soaked in a solution containing phenylalanine and ATP in the presence of Mn(2+) ions. The first step of the aminoacylation reaction proceeds within the crystals, resulting in Phe-AMP formation at the active site. Specific recognition of the phenylalanine portion of the Phe-AMP is achieved by interactions of the phenyl ring of Phe-AMP with two neighbouring residues, Phealpha258 and Phealpha260. No manganese ions were observed within the active site; their role in the formation of the transition state may be assigned to a number of polar residues and water molecules. In the anomalous Fourier difference map, a divalent metal ion was detected at the interface of the alpha- and beta-subunits at a short distance from motif 3 residues participating in the substrate binding. A sulfate ion, which was identified on the protein surface, may mediate the interactions of PheRS with DNA. Visible conformational changes were detected in the active-site area adjacent to the position of the Phe-AMP, compared with the structure of PheRS complexed with a synthetic adenylate analogue (phenylalaninyl-adenylate). Based on the known structures of the substrate-free enzyme and its complexes with various ligands, a general scheme for the phenylalanylation mechanism is proposed.

A peculiarity of the reaction of tRNA aminoacylation catalyzed by phenylalanyl-tRNA synthetase from the extreme thermophile Thermus thermophilus

It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation reaction catalyzed by Thermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two bound phenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quite low: the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. The thermophilic synthetase can aminoacylate twice not only T. thermophilus tRNAPhe but also Escherichia coli tRNAPhe and E. coli tRNAPhe transcript, indicating that the presence of modified nucleotides is not necessary for tRNAPhe overcharging. Bisphenylalanyl-tRNA is stable in acidic solution, but it decomposes in alkaline medium yielding finally tRNA and free phenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate as from monophenylalanyl-tRNA. In the presence of the enzyme the rate of bisphenylalanyl-tRNA deacylation increases. Aminoacylated tRNAPhe isolated from T. thermophilus living cells was observed to contain no detectable bisphenylalanyl-tRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed.

Phenylalanyl-tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNAPhe

Phenylalanyl‐tRNA synthetase from the extreme thermophilic bacterium Thermus thermophilus can incorporate more than one molecule of phenylalanine into the tRNAPhe. It is shown that the ‘hyperaminoncylated’ tRNAPPhe is the bis‐2′,3′‐O‐phenylalanyl‐tRNAPhe, and its formation is typical for the thermophilic enzyme but does not occur for E. coli phenylalanyl‐tRNA synthetase under the same conditions.

Determination of tRNAPhe nucleotides contacting the subunits of Thermus thermophilus phenylalanyl-tRNA synthetase by photoaffinity crosslinking

The nucleotides of tRNA(Phe) interacting with the subunits of Thermus thermophilus phenylalanyl-tRNA synthetase (the alpha(2)beta(2) heterotetramer) have been determined by photoaffinity crosslinking of randomly s(4)U-monosubstituted tRNA(Phe) transcripts which retain aminoacylation parameters closely similar to those of the native tRNA(Phe). The thiolated transcripts have been fractionated by affinity electrophoresis and separately crosslinked to the enzyme. Sites of crosslinking to the beta subunit have been identified at positions 33 and 39 and crosslinking sites to the alpha subunit have been localized at positions 20, 45 and 47, using alkaline hydrolysis analysis of the crosslinked proteinase K-treated tRNAs. An additional crosslink to the beta subunit, not identified in the full-length crosslinked tRNAs, has been deduced to occur at position 12, based on the analysis of an unusual (fast migrating) crosslinked product. Nucleotide s(4)U8 of native tRNA(Phe) has been shown to form a minor crosslink to the alpha subunit. Four of the seven crosslinking sites, namely nucleotides 8, 12, 20 and 39, are among those shown to be protected against cleavage by iodine in footprinting experiments; in contrast, only nucleotide 12 is among the contact sites defined in the crystal structure. The data of independent biochemical approaches strongly suggest conformational flexibility of the complex under functional conditions, thus reflecting the importance of macromolecular dynamics for the interaction.

Comparative study of subunits of phenylalanyl-tRNA synthetase from Escherichia coli and Thermus thermophilus

FPLC separation of α‐ and β‐subunits of phenylalanyl‐tRNA synthetases from E. coli MRE‐600 and Thermus thermophilus HB8 has been carried out in the presence of urea. Native α‐subunits of both enzymes were primarily α2‐dimers and tended to aggregate. Most E. coli enzyme β‐subunits were monomeric and only a small fraction was represented by β2‐dimers. All thermophilic β‐subunits were β‐dimers. It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNAPhe aminoacylation. For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined. The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding β‐subunits. α‐Subunits displayed a distinctly different pH dependence of the surface charge. A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed.