Recombinant mammalian tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on beta-tubulin through a random sequential pathway.
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 beta-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 beta-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 beta-tubulin through a random sequential pathway.
Mukai
Masahiro
M
Mitsubishi Kagaku Institute of Life Sciences (MITILS), Minamiooya, Machida, Tokyo 194-8511, Japan.
Ikegami
Koji
K
Sugiura
Yuki
Y
Takeshita
Kouhei
K
Nakagawa
Atsushi
A
Setou
Mitsutoshi
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D005971:Glutamates / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D010453:Peptide Synthases / Q000737:chemistry / Q000502:physiology*; D011099:Polyglutamic Acid / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*
D005971:Glutamates; D011994:Recombinant Proteins; D014404:Tubulin; D011099:Polyglutamic Acid; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C112698:tubulin polyglutamylase