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

27 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

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Recombinant mammalian Tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on ß-Tubulin through a random sequential pathway'. Together they form a unique fingerprint.

  • Cite this