Recombinant mammalian Tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on ß-Tubulin through a random sequential pathway

Masahiro Mukai, Koji Ikegami, Yuki Sugiura, Kouhei Takeshita, Atsushi Nakagawa, Mitsutoshi Setou

研究成果: Article

27 引用 (Scopus)

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Tubulins undergo unique post-translational modifications, such as tyrosination, polyglutamylation, and polyglycylation. These modifications are performed by members of a protein family, the tubulin tyrosine ligase (TTL)-like (TTLL) family, which is characterized by the presence of a highly conserved TTL domain. We and others have recently identified tubulin polyglutamylases in the TTLL family [Janke, C, et al. (2005) Science 308, 1758-1762; Ikegami, K., et al. (2006) J. Biol. Chem. 281, 30707-30716; van Dijk, J., et al. (2007) Mol. Cell 26, 437-448]. Previously, we identified TTLL7 as a β-tubulin-selective polyglutamylase. However, there is controversy over whether TTLL7 functions as an initiase, elongase, or both in polyglutamylation. In this report, we investigate the polyglutamylation reaction by TTLL7 by employing a recombinant enzyme and in vitro reaction. Two-dimensional electrophoresis and tandem mass spectrometry showed that TTLL7 performed both the initiation and elongation of polyglutamylation on β-tubulin. Recombinant TTLL7 performed with a maximal and specific activity to polymerized tubulin at a neutral pH and a lower salt concentration. The initial rate and inhibitor analyses revealed that the mechanism of binding of three substrates, glutamate, ATP, and tubulin, to the enzyme was a random sequential pathway. Our findings provide evidence that mammalian TTLL7 performs both initiation and elongation in the polyglutamylation reaction on β-tubulin through a random sequential pathway.

元の言語English
ページ(範囲)1084-1093
ページ数10
ジャーナルBiochemistry
48
発行部数5
DOI
出版物ステータスPublished - 2009 2 10

ASJC Scopus subject areas

  • Biochemistry

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