Unexpected Preference of the E. coli Translation System for the Ester Bond during Incorporation of Backbone-Elongated Substrates

There have been recent advances in the ribosomal synthesis of various molecules composed of nonnatural ribosomal substrates. However, the ribosome has strict limitations on substrates with elongated backbones. Here, we show an unexpected loophole in the E. coli translation system, based on a remarka...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2007-05, Vol.129 (19), p.6180-6186
Main Authors: Sando, Shinsuke, Abe, Kenji, Sato, Nobuhiko, Shibata, Toshihiro, Mizusawa, Keigo, Aoyama, Yasuhiro
Format: Article
Language:English
Subjects:
Amino Acids - chemistry
Blotting, Western
Electrophoresis, Polyacrylamide Gel
Escherichia coli - genetics
Escherichia coli - metabolism
Esters
Lactic Acid - analogs & derivatives
Lactic Acid - chemistry
Oligopeptides - biosynthesis
Protein Biosynthesis
Ribosomes - metabolism
RNA, Bacterial - metabolism
RNA, Transfer, Amino Acyl - metabolism
Substrate Specificity
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Summary:There have been recent advances in the ribosomal synthesis of various molecules composed of nonnatural ribosomal substrates. However, the ribosome has strict limitations on substrates with elongated backbones. Here, we show an unexpected loophole in the E. coli translation system, based on a remarkable disparity in its selectivity for β-amino/hydroxy acids. We challenged β-hydroxypropionic acid (β-HPA), which is less nucleophilic than β-amino acids but free from protonation, to produce a new repertoire of ribosome-compatible but main-chain-elongated substrates. PAGE analysis and mass-coupled S-tag assays of amber suppression experiments using yeast suppressor tRNAPhe CUA confirmed the actual incorporation of β-HPA into proteins/oligopeptides. We investigated the side-chain effects of β-HPA and found that the side chain at position α and R stereochemistry of the β-substrate is preferred and even notably enhances the efficiency of incorporation as compared to the parent substrate. These results indicate that the E. coli translation machinery can utilize main-chain-elongated substrates if the pK a of the substrate is appropriately chosen.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja068033n