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

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1084-1093
Number of pages10
JournalBiochemistry
Volume48
Issue number5
DOIs
Publication statusPublished - 2009 Feb 10
Externally publishedYes

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Tubulin
Elongation
Transistor transistor logic circuits
Enzymes
Post Translational Protein Processing
Tandem Mass Spectrometry
Electrophoresis
Glutamic Acid
Salts
Adenosine Triphosphate
Mass spectrometry
tubulin polyglutamylase
Substrates
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Recombinant mammalian Tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on ß-Tubulin through a random sequential pathway. / Mukai, Masahiro; Ikegami, Koji; Sugiura, Yuki; Takeshita, Kouhei; Nakagawa, Atsushi; Setou, Mitsutoshi.

In: Biochemistry, Vol. 48, No. 5, 10.02.2009, p. 1084-1093.

Research output: Contribution to journalArticle

Mukai, Masahiro ; Ikegami, Koji ; Sugiura, Yuki ; Takeshita, Kouhei ; Nakagawa, Atsushi ; Setou, Mitsutoshi. / Recombinant mammalian Tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on ß-Tubulin through a random sequential pathway. In: Biochemistry. 2009 ; Vol. 48, No. 5. pp. 1084-1093.
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