Abstract
Eubacterial leucyl/phenylalanyl-tRNA protein transferase (LF-transferase) catalyses peptide-bond formation by using Leu-tRNALeu (or Phe-tRNAPhe) and an amino-terminal Arg (or Lys) of a protein, as donor and acceptor substrates, respectively. However, the catalytic mechanism of peptide-bond formation by LF-transferase remained obscure. Here we determine the structures of complexes of LF-transferase and phenylalanyl adenosine, with and without a short peptide bearing an N-terminal Arg. Combining the two separate structures into one structure as well as mutation studies reveal the mechanism for peptide-bond formation by LF-transferase. The electron relay from Asp 186 to Gln 188 helps Gln 188 to attract a proton from the α-amino group of the N-terminal Arg of the acceptor peptide. This generates the attacking nucleophile for the carbonyl carbon of the aminoacyl bond of the aminoacyl-tRNA, thus facilitating peptide-bond formation. The protein-based mechanism for peptide-bond formation by LF-transferase is similar to the reverse reaction of the acylation step observed in the peptide hydrolysis reaction by serine proteases.
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Acknowledgements
We thank S. Fukai, T. Numata, T. Suzuki and H. Hori for valuable and critical comments, and suggestions for this manuscript. We thank the beam-line staffs at BL-5A, BL-17A and AR-NW 12A of KEK (Tsukuba, Japan) for technical help during data collection, and A. Hamada for technical assistance. This work was supported in part by grants from JSPS, MEXT and the Kurata Memorial Hitachi Science and Technology Foundation (K.T.).
Author Contributions K.T. purified and crystallized the proteins, and K.W., Y.T. and K.T. collected the data and determined the structures. K.S. assisted with the structural analysis, K.W. and K.T. carried out biochemical and mass analyses, Y.S. assisted with the mass analysis, and N.O. and T.W. synthesized analogues. K.W., Y.T. and K.T. wrote the paper. All authors discussed the results and commented on the manuscript.
Coordinates and structure factors have been deposited in the Protein Data Bank, under the accession codes 2Z3K, 2Z3L, 2Z3M, 2Z3N, 2Z3O and 2Z3P.
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Watanabe, K., Toh, Y., Suto, K. et al. Protein-based peptide-bond formation by aminoacyl-tRNA protein transferase. Nature 449, 867–871 (2007). https://doi.org/10.1038/nature06167
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DOI: https://doi.org/10.1038/nature06167
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