Abstract
Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNALeu and phenylalanyl-tRNAPhe as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.
Keywords: Aminoacyl-tRNA protein transferase, L/F transferase, N-end rule, puromycin, quantitative mass spectrometry.
Protein & Peptide Letters
Title:Probing the Leucyl/Phenylalanyl tRNA Protein Transferase Active Site with tRNA Substrate Analogues
Volume: 21 Issue: 7
Author(s): Angela Wai Shan Fung, H. Alexander Ebhardt, Kollappillil S. Krishnakumar, Jack Moore, Zhizhong Xu, Peter Strazewski and Richard P. Fahlman
Affiliation:
Keywords: Aminoacyl-tRNA protein transferase, L/F transferase, N-end rule, puromycin, quantitative mass spectrometry.
Abstract: Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNALeu and phenylalanyl-tRNAPhe as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.
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Fung Wai Shan Angela, Ebhardt Alexander H., Krishnakumar S. Kollappillil, Moore Jack, Xu Zhizhong, Strazewski Peter and Fahlman P. Richard, Probing the Leucyl/Phenylalanyl tRNA Protein Transferase Active Site with tRNA Substrate Analogues, Protein & Peptide Letters 2014; 21 (7) . https://dx.doi.org/10.2174/0929866521666140212110639
DOI https://dx.doi.org/10.2174/0929866521666140212110639 |
Print ISSN 0929-8665 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5305 |
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