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

Angela Wai Shan Fung, H Alexander Ebhardt, Kollappillil S Krishnakumar, Jack Moore, Zhizhong Xu, Peter Strazewski, Richard P Fahlman

Protein and peptide letters 2014 Jul

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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.

Citation

Angela Wai Shan Fung, H Alexander Ebhardt, Kollappillil S Krishnakumar, Jack Moore, Zhizhong Xu, Peter Strazewski, Richard P Fahlman. Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues. Protein and peptide letters. 2014 Jul;21(7):603-14