Abstract
Polypeptides containing β-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of β-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of β3-Met by Escherichia coli methionyl-tRNA synthetase (MetRS). We show that MetRS binds β3-Met with a 24-fold lower affinity but catalyzes the esterification of the non-canonical amino acid onto tRNA with a rate lowered by three orders of magnitude. Accurate measurements of the catalytic parameters required careful consideration of the presence of contaminating α-Met in β3-Met commercial samples. The 1.45 Å crystal structure of the MetRS: β3-Met complex shows that β3-Met binds the enzyme essentially like α-Met, but the carboxylate moiety is mobile and not adequately positioned to react with ATP for aminoacyl adenylate formation. This study provides structural and biochemical bases for engineering MetRS with improved β3-Met aminoacylation capabilities.
Highlights
The design of polypeptides having enhanced or novel properties is a major challenge for synthetic biology
Prior to studying activation of β3-Met by methionyltRNA synthetase (MetRS), we used Liquid chromatography/ high-resolution mass spectrometry (LC-HRMS) experiments to search for the presence of α-Met in the β3-Met sample
BOC-L-methionine was used as a reactant during the synthesis of β3-Met, as communicated by the company from which the compound was purchased
Summary
The design of polypeptides having enhanced or novel properties is a major challenge for synthetic biology. A key requirement for in vivo approaches is to ensure the availability of tRNA aminoacylated with the non-standard amino acid. In some cases, this has been done using mutated aminoacyl-tRNA synthetases (aaRS) able to aminoacylate a wild-type tRNA. For site-specific incorporation, the non-standard amino acid is generally encoded by a non-sense or a frameshift codon (Chin, 2017; Liu and Schultz, 2010) In this case, an orthogonal tRNA, not recognized by any endogeneous aaRS, is used together with an orthogonal aaRS, unable to aminoacylate any endogeneous tRNA (Wang et al, 2006). These methods are based on RNA catalysts named flexizymes (Murakami et al, 2003, 2006; Ohuchi et al, 2007) and have virtually no limitation of acid donor substrates
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