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Journal of Biochemistry Advance Access originally published online on April 22, 2009
Journal of Biochemistry 2009 146(2):241-250; doi:10.1093/jb/mvp063
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Analyses of Conditions for KMSSS Loop in Tyrosyl-tRNA Synthetase by Building a Mutant Library

Shunsuke Kamijo*, Akihiko Fujii, Kenji Onodera and Kenichi Wakabayashi

The University of Tokyo, Institute of Industrial Science, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan

*To whom correspondence should be addressed. Tel: +81-3-5452-6272, Fax: +81-3-5452-6274, E-mail: kamijo{at}iis.u-tokyo.ac.jp

Received December 7, 2008; Accepted April 6, 2009


  Abstract

The KMSKS motif is the ATP binding motif for aminoacylation process of class I aminoacyl-tRNA synthetases. Although researches based on natural proteins inform us about the contribution of natural amino acid sequences for the catalysis, they have difficulties in discussing the other alternative sequences and prohibited sequences for the motif to maintain the catalytic ability. In order to reveal such the conditions for the alternative and prohibited sequences, it is important to investigate a library of various mutants for the motif. For that purpose, we build a library of more than 200 mutants substituting the KMSSS loop, Lys204-Met205-Ser206-Ser207-Ser208, in tyrosyl-tRNA synthetase of Methanococcus jannaschii, and their catalytic abilities were examined by the Amber suppression method. Mutants of K204R and K204N still maintained catalytic abilities to a certain extent. On the other hand, a variety of alternative sequences for Ser206-Ser207-Ser208 were obtained, and some of those did not include either Ser or Thr, which were regarded as necessary residues in the KMSKS motif in previous works. In this article, catalytic activity of all the mutants are represented in detail and some suggestions for the condition of the motif are discussed.

Key Words: amino-acyl tRNA Synthetase, ATP binding motif, KMSKS loop, amber suppression, mutational analysis


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