Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues

• Published in: Protein and peptide letters Vol. 21; no. 7; p. 603
• Main Authors: Fung, Angela Wai Shan, Ebhardt, H Alexander, Krishnakumar, Kollappillil S, Moore, Jack, Xu, Zhizhong, Strazewski, Peter, Fahlman, Richard P
• Format: Journal Article
• Language: English
• Published: Netherlands 01.07.2014
• Subjects: Aminoacyltransferases - antagonists & inhibitors; Aminoacyltransferases - chemistry; Aminoacyltransferases - metabolism; Catalytic Domain; Crystallography, X-Ray; Escherichia coli Proteins; Mass Spectrometry; Models, Molecular; Mutation; Protein Binding; Puromycin - pharmacology; Recombinant Fusion Proteins; RNA, Transfer, Leu - chemistry; RNA, Transfer, Leu - metabolism; RNA, Transfer, Phe - chemistry; RNA, Transfer, Phe - metabolism
• ISSN: 1875-5305
• DOI: 10.2174/0929866521666140212110639

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-tRNA(Leu) and phenylalanyl-tRNA(Phe) 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.