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Aminoacyl-tRNA synthetases are responsible for attaching amino acid residues to the tRNA 3′-end. The two classes of synthetases approach tRNA as mirror images, with opposite but symmetrical stereochemistries that allow the class I enzymes to attach amino acid residues to the 2′-hydroxyl group of the terminal ribose, whereas, the class II enzymes attach amino acid residues to the 3′-hydroxyl group. However, we show here that the attachment of cysteine to tRNACys by the class I cysteinyl-tRNA synthetase (CysRS) is flexible; the enzyme is capable of using either the 2′ or 3′-hydroxyl group as the attachment site. The molecular basis for this flexibility was investigated. Introduction of the nucleotide U73 of tRNACys into tRNAVal was found to confer the flexibility. While valylation of the wild-type tRNAVal by the class I ValRS was strictly dependent on the terminal 2′-hydroxyl group, that of the U73 mutant of tRNAVal occurred at either the 2′ or 3′-hydroxyl group. Thus, the single nucleotide U73 of tRNA has the ability to break the stereo barrier of amino acid attachment to tRNA, by mobilizing the 2′ and 3′-hydroxyl groups of A76 in flexible geometry with respect to the tRNA acceptor stem. [Copyright &y& Elsevier]

Cysteinyl‐tRNA synthetase (CysRS) is highly specific for synthesis of cysteinyl adenylate, yet does not possess the amino acid editing activity characteristic of many other tRNA synthetases. To elucidate how CysRS is able to distinguish cysteine from non‐cognate amino acids, crystal structures of the Escherichia coli enzyme were determined in apo and cysteine‐bound states. The structures reveal that the substrate cysteine thiolate forms a single direct interaction with a zinc ion bound at the base of the active site cleft, in a trigonal bipyramidal geometry together with four highly conserved protein side chains. Cysteine binding induces movement of the zinc ion towards substrate, as well as flipping of the conserved Trp205 indole ring to pack on the thiol side chain. The imidazole groups of five conserved histidines lie adjacent to the zinc ion, forming a unique arrangement suggestive of functional significance. Thus, amino acid discrimination without editing arises most directly from the favorable zinc–thiolate interaction, which is not possible for non‐cognate substrates. Additional selectivity may be generated during the induced‐fit conformational changes that help assemble the active site. [ABSTRACT FROM AUTHOR]

The article focuses on the study on the mechanism of making cyclodipeptides from aminoacyl-transfer RNA (aa-tRNA). It suggests that cyclodipeptide synthases (CDPSs) can hijack aa-tRNAs to make cyclodipeptides. It shows that the biosynthesis of the CDP is expected to have an impact on human health. It indicates that the protein sequence profile analysis suggests that CDPSs may be related to the aa-tRNA synthesizing aminoacyl-tRNA synthetases (aaRSs).

Abstract: Aminoacyl-tRNA synthetases are an ancient class of enzymes responsible for the matching of amino acids with anticodon sequences of tRNAs. Eukaryotic tRNA synthetases are often larger than their bacterial counterparts, and several mammalian enzymes use the additional domains to facilitate assembly into a multi-synthetase complex. Human cysteinyl-tRNA synthetase (CysRS) does not associate with the multi-synthetase complex, yet contains a eukaryotic-specific C-terminal extension that follows the tRNA anticodon-binding domain. Here we show by mutational and kinetic analysis that the C-terminal extension of human CysRS is used to selectively improve recognition and binding of the anticodon sequence, such that the specificity of anticodon recognition by human CysRS is higher than that of its bacterial counterparts. However, the improved anticodon recognition is achieved at the expense of a significantly slower rate in the aminoacylation reaction, suggesting a previously unrecognized kinetic quality control mechanism. This kinetic quality control reflects an evolutionary adaptation of some tRNA synthetases to improve the anticodon specificity of tRNA aminoacylation from bacteria to humans, possibly to accommodate concomitant changes in codon usage. [Copyright &y& Elsevier]

Abstract: Aminoacyl-tRNA synthetases are divided into two classes based on both functional and structural criteria. Distinctions between the classes have heretofore been based on general features, such as the position of aminoacylation on the 3′-terminal tRNA ribose, and the topology and tRNA-binding orientation of the active-site protein fold. Here we show instead that transient burst kinetics provides a distinct mechanistic signature dividing the two classes of tRNA synthetases, and that this distinction has significant downstream effects on protein synthesis. Steady-state and transient kinetic analyses of class I CysRS and ValRS, and class II AlaRS and ProRS, reveal that class I tRNA synthetases are rate-limited by release of aminoacyl-tRNA, while class II synthetases are limited by a step prior to aminoacyl transfer. The tight aminoacyl-tRNA product binding by class I enzymes correlates with the ability of EF-Tu to form a ternary complex with class I but not class II synthetases, and the further capacity of this protein to enhance the rate of aminoacylation by class I synthetases. These results emphasize that the distinct mechanistic signatures of class I versus class II tRNA synthetases ensure rapid turnover of aminoacyl-tRNAs during protein synthesis. [Copyright &y& Elsevier]

Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves a high level of amino acid specificity without an editing reaction. The crystal structure of CysRS bound to substrate cysteine suggested that direct thiol coordination to a tightly bound zinc ion at the base of the active site is the primary determinant of selectivity against non-cognate amino acids. This hypothesis has now been supported by spectroscopic studies of cobalt-substituted CysRS. Binding of cysteine, but not non-cognate amino acids, induces high absorption in the ligand-to-metal charge transfer region, providing evidence for formation of a metal–thiolate bond. In addition, mutations in the zinc ligands alter the absorption spectrum without reducing the discrimination against non-cognate amino acids. These results argue strongly for a major role for the zinc ion in amino acid discrimination by CysRS, where the tight zinc–thiolate interaction and the strict structural geometry of the metal ion are sufficient to reject serine by more than 20,000-fold at the binding step. [Copyright &y& Elsevier]