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 that catalyzes serine activation and its transfer to cognate tRNA(Ser). Previous biochemical and structural studies have revealed that bacterial- and methanogenic-type SerRSs employ different strategies of substrate recognition. In addition to other idiosyncratic features, such as the active site zinc ion and the unique fold of the N-terminal tRNA-binding domain, methanogenic-type SerRS is, in comparison with bacterial homologues, characterized by a notable shortening of the motif 2 loop. Mutational analysis of Methanosarcina barkeri SerRS (mMbSerRS) was undertaken to identify the active site residues that ensure the specificity of amino acid and tRNA 3'-end recognition. Residues predicted to contribute to the amino acid specificity were selected for mutation according to the crystal structure of mMbSerRS complexed with its cognate aminoacyl-adenylate, whereas those involved in binding of the tRNA 3'-end were identified and mutagenized on the basis of modeling the mMbSerRS:tRNA complex. Although mMbSerRSs variants with an altered serine-binding pocket (W396A, N435A, S437A) were more sensitive to inhibition by threonine and cysteine, none of the mutants was able to activate noncognate amino acids to greater extent than the wild-type enzyme. In vitro kinetics results also suggest that conformational changes in the motif 2 loop are required for efficient serylation.
Bilokapic
Silvija
S
Department of Chemistry, Faculty of Science, University of Zagreb, Croatia.
Rokov Plavec
Jasmina
J
Ban
Nenad
N
Weygand-Durasevic
Ivana
I
D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008697:Methane / Q000737:chemistry; D017021:Methanosarcina barkeri / Q000201:enzymology; D008958:Models, Molecular; D008968:Molecular Conformation; D012343:RNA, Transfer / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D012714:Serine-tRNA Ligase / Q000737:chemistry*; D013379:Substrate Specificity
D000596:Amino Acids; D012694:Serine; D012343:RNA, Transfer; D012714:Serine-tRNA Ligase; D008274:Magnesium; D008697:Methane