engleski

Structural flexibility of the methanogenic-type seryl-tRNA synthetase active site and its implication for specific substrate recognition

Seryl-tRNA synthetase (SerRS) is a class II aminoacyl-tRNA synthetase (aaRS) that catalyzes serine activation and its transfer to cognate tRNASer. Previous biochemical and structural studies revealed that bacterial- and methanogenic- type SerRSs employ different strategies of substrate recognition. Besides other idiosyncratic features, such as the active site zinc ion and unique fold of the N-terminal tRNA binding domain, the methanogenic-type SerRS is, in comparison with bacterial homologues, characterized by a notable shortening of the motif 2 loop. Mutational analysis of the Methanosarcina barkeri SerRS (mMbSerRS) was undertaken with the goal of identifying the active site residues that ensure the specificity of amino acid and tRNA 3'-end recognition. The residues predicted to contribute to amino acid specificity were selected for mutations according to the crystal structure of mMbSerRS complexed with its cognate aminoacyl- adenylate, while those involved in the binding of the 3'-end of tRNA were identified and mutagenised on the basis of modeling the mMbSerRS:tRNA complex. Although the mMbSerRSs variants with altered serine binding pocket (W396A, N435A, S437A) were more sensitive to the inhibition by threonine and cysteine, none of the mutants were able to activate non-cognate amino acids to higher extent than the wild-type enzyme. In vitro kinetic results also suggest that conformational changes of motif 2 loop are required for efficient serylation.

conformational flexibility ; motif 2 loop ; seryl-tRNA synthetase ; specificity of substrate recognition ; synthetase:tRNA model

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