Information on EC 6.3.2.61 - tubulin-glutamate ligase

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tubulin tyrosine ligase like

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763301

tubulin tyrosine ligase-like

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tubulin tyrosine ligase-like 1

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catalytic subunit

762893

tubulin tyrosine ligase-like protein 1

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763639, 763743

tubulin-tyrosine ligase-like protein

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additional information

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tubulin glutamylases belong to the family of TTL domain-carrying proteins named TTLL proteins

ATP + [tubulin]-(alpha-L-glutamyl-gamma-L-glutamyl)-L-glutamate + n L-glutamate = [tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

(1c)

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ATP + [tubulin]-(gamma-L-glutamyl)-L-glutamate + L-glutamate = [tubulin]-(alpha-L-glutamyl-gamma-L-glutamyl)-L-glutamate + ADP + phosphate

(1b)

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ATP + [tubulin]-L-glutamate + L-glutamate = [tubulin]-(gamma-L-glutamyl)-L-glutamate + ADP + phosphate

n ATP + [tubulin]-L-glutamate + n L-glutamate = [tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

overall reaction

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[tubulin]-L-glutamate:L-glutamate ligase (ADP-forming)

The eukaryotic tubulin proteins, which polymerize into microtubules, are highly modified by the addition of side-chains. The polyglutamylation reaction catalysed by this group of enzymes consists of two biochemically distinct steps: initiation and elongation. Initiation comprises the formation of an isopeptide bond with the gamma-carboxyl group of the glutamate acceptor site in a glutamate-rich C-terminal region of tubulin, whereas elongation consists of the addition of glutamate residues linked by regular peptide bonds to the gamma-linked residue. This entry describes enzymes that act on both alpha- and beta-tubulins.

ATP + acidic nuclear phosphoprotein 32 family, member A + L-glutamate

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ATP + acidic nuclear phosphoprotein 32 family, member B + L-glutamate

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ATP + alpha tubulin + L-glutamate

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4 entries

ATP + alpha-tubulin + L-glutamate

L-glutamyl-alpha-tubulin + ADP + phosphate

ATP + B23 nucleophosmin + L-glutamate

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ATP + EB1 protein + L-glutamate

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Substrates: very low activity with isoform TTLL4
Products: -

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ATP + microtubule + L-glutamate

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ATP + NF45 protein + L-glutamate

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ATP + NPN2 protein + L-glutamate

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Substrates: high activity with isoform TTLL4
Products: -

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ATP + NPN3 protein + L-glutamate

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Substrates: very low activity with isoform TTLL4
Products: -

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ATP + nucleosome assembly protein 1-like 1 + L-glutamate

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ATP + nucleosome assembly protein 1-like 4 + L-glutamate

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ATP + protein SET + L-glutamate

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ATP + [alpha-tubulin]-L-glutamate + L-glutamate

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Substrates: the enzyme catalyzes the MgATP-dependent addition of L-glutamate onto tubulin subunits. The enzyme is active with alpha- and beta-tubulin, respectively. Microtubules are much better substrates than unpolymerized tubulin, and the reaction is very specific for glutamate, other amino acids or glutamate analogues not being substrates. Moreover, glutamyl units are added sequentially onto tubulin, leading to progressive elongation of the polyglutamyl side chains. Side chains of one to six or seven glutamyl units are obtained with microtubules, whereas much longer side chains (up to 15-20 units) are formed with unpolymerized tubulin
Products: -

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ATP + [alpha/beta-tubulin]-(alpha-L-glutamyl-gamma-L-glutamyl)-L-glutamate + n L-glutamate

[alpha/beta-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

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Substrates: (1c)
Products: -

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ATP + [alpha/beta-tubulin]-(gamma-L-glutamyl)-L-glutamate + L-glutamate

[alpha/beta-tubulin]-(alpha-L-glutamyl-gamma-L-glutamyl)-L-glutamate + ADP + phosphate

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Substrates: (1b)
Products: -

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ATP + [alpha/beta-tubulin]-L-glutamate + L-glutamate

[alpha/beta-tubulin]-(gamma-L-glutamyl)-L-glutamate + ADP + phosphate

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Substrates: (1a)
Products: -

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ATP + [beta-tubulin]-L-glutamate + L-glutamate

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Substrates: the enzyme catalyzes the MgATP-dependent addition of L-glutamate onto tubulin subunits. The enzyme is active with alpha- and beta-tubulin, respectively. Microtubules are much better substrates than unpolymerized tubulin, and the reaction is very specific for glutamate, other amino acids or glutamate analogues not being substrates. Moreover, glutamyl units are added sequentially onto tubulin, leading to progressive elongation of the polyglutamyl side chains. Side chains of one to six or seven glutamyl units are obtained with microtubules, whereas much longer side chains (up to 15-20 units) are formed with unpolymerized tubulin
Products: -

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n ATP + [alpha-tubulin]-(alpha-L-glutamyl-gamma-L-glutamyl)-L-glutamate + n L-glutamate

[alpha-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

Q91V51, Q8CHB8, A4Q9E8, A4Q9F4, A4Q9F6

751550

Substrates: no initiating activity, high elongation activity. The enzyme modifies both alpha- and beta-tubulin and shows a preference towards alpha-tubulin
Products: -

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n ATP + [alpha-tubulin]-L-glutamate + n L-glutamate

[alpha-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

5 entries

n ATP + [alpha/beta-tubulin]-L-glutamate + n L-glutamate

[alpha/beta-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

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Substrates: overall reaction. The enzyme incorporates glutamic acid preferentially into the more acidic variants of both alpha- and beta-tubulins
Products: -

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n ATP + [beta-tubulin]-(alpha-L-glutamyl-gamma-L-glutamyl)-L-glutamate + n L-glutamate

[beta-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

Q91V51, Q8CHB8, A4Q9E8, A4Q9F4, A4Q9F6

751550

Substrates: no initiating activity, high elongation activity. The enzyme modifies both alpha- and beta-tubulin and shows a preference towards alpha-tubulin
Products: -

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n ATP + [beta-tubulin]-L-glutamate + n L-glutamate

[beta-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

4 entries

n ATP + [modified porcine brain tubulin]-L-glutamate + n L-glutamate

[modified porcine brain tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

Substrates: -
Products: -

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n ATP + [tubulin]-L-glutamate + n L-glutamate

[tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

5 entries

n ATP + [unmodified human tubulin]-L-glutamate + n L-glutamate

[unmodified human tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

Substrates: -
Products: -

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additional information

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15 entries

ATP + alpha-tubulin + L-glutamate

L-glutamyl-alpha-tubulin + ADP + phosphate

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Substrates: glutamylation on alpha-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level
Products: -

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ATP + [alpha/beta-tubulin]-(alpha-L-glutamyl-gamma-L-glutamyl)-L-glutamate + n L-glutamate

[alpha/beta-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

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Substrates: (1c)
Products: -

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ATP + [alpha/beta-tubulin]-(gamma-L-glutamyl)-L-glutamate + L-glutamate

[alpha/beta-tubulin]-(alpha-L-glutamyl-gamma-L-glutamyl)-L-glutamate + ADP + phosphate

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Substrates: (1b)
Products: -

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ATP + [alpha/beta-tubulin]-L-glutamate + L-glutamate

[alpha/beta-tubulin]-(gamma-L-glutamyl)-L-glutamate + ADP + phosphate

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Substrates: (1a)
Products: -

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n ATP + [alpha-tubulin]-L-glutamate + n L-glutamate

[alpha-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

n ATP + [alpha/beta-tubulin]-L-glutamate + n L-glutamate

[alpha/beta-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

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Substrates: overall reaction. The enzyme incorporates glutamic acid preferentially into the more acidic variants of both alpha- and beta-tubulins
Products: -

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n ATP + [beta-tubulin]-L-glutamate + n L-glutamate

[beta-tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

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749873, 763304, 763305

Substrates: overall reaction
Products: -

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n ATP + [tubulin]-L-glutamate + n L-glutamate

[tubulin]-(gamma-(poly-alpha-L-glutamyl)-L-glutamyl)-L-glutamate + n ADP + n phosphate

5 entries

additional information

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Substrates: Ttll1p and Ttll9p are tubulin tyrosine ligase domain proteins, that act as alpha-tubulin-preferring glutamyl ligase enzymes, TTLL1- and TLLL9-type enzymes are highly conserved but are absent from higher plants and fungi, overview
Products: -

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ATP

Mg2+

8 entries

ATP

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90% decrease of activity at 10 mM ATP

citrate

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about 90% inhibition at 10 mM

KCl

Mg2+

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75% decrease of activity at 20 mM Mg2+

Na2SO4

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90% inhibition at 20 mM

NaCl

Sodium acetate

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100 mM, 50% onhibition

Sodium citrate

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90% inhibition at 10 mM

Sodium sulfate

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about 90% inhibition at 20 mM

glycerol

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20% (v/v) leads to 10-15% increase in glutamylation activity

0.1

ATP

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30°C, pH 8.7

0.8 - 3

L-glutamate

3 entries

0.0003

[alpha/beta-tubulin]-L-glutamate

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at pH 9.0, temperature not specified in the publication

0.00809

[modified porcine brain tubulin]-L-glutamate

pH and temperature not specified in the publication

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0.00773

[unmodified human tubulin]-L-glutamate

pH and temperature not specified in the publication

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0.0032

[modified porcine brain tubulin]-L-glutamate

pH and temperature not specified in the publication

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0.0037

[unmodified human tubulin]-L-glutamate

pH and temperature not specified in the publication

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8.3 - 8.9

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9

additional information

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the high pH optimum observed at low glutamate concentrations is a result of the free amine of glutamate (pK = 9.7 at 25°C) being the substrate for the reaction

6.8 - 8.9

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the enzyme activity increases markedly with the pH going from 6.8 to 8.3, with a large optimum between 8.3 and 8.9, and then decreases sharply. At physiological pH, the activity is 15% of the maximal level

7.5 - 9.2

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pH 7.5: about 45% of maximal activity, pH 9.2: about 80% of maximal activity. When the glutamate concentration is raised to 6 mM, the activity is less dependent on pH, decreasing by only 40% between pH 8.7 and 6.8

30

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assay at

Chlamydomonas reinhardtii

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763459

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Chlamydomonas reinhardtii CC-124

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763459

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763025, 763303

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Danio rerio

wild type TuAB and CD-41 GFP zebrafish lines

751028

A8CVX7

UniProt

Drosophila melanogaster

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retinal pigment epithelium cell

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gut

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Drosophila melanogaster

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lowest expression

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5874

Highest Expressing Human Cell Lines

Filter by:

Cell Line Links

Gene Links

malfunction

9 entries

physiological function

360000

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multimer

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x * 8000 + x * 50000 + x * 32000, SDS-PAGE

763305

wild type and mutant enzyme C179A/Q180R/H362I, in complex with ATP and inhibitors, hanging dop vapor diffusion method, using 0.2 M ammonium sulfate and 10-15% (w/v) PEG 3350

C179A/Q180R/H362I

the mutant no longer discriminates between heterogenous glutamylated brain microtubules and unmodified microtubules, as both the kcat and Km are similar with these substrates. The triple-mutant has strong preference for glutamylating an unmodified alpha1B peptide

E439A

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inactive mutant of isoform TTLL6

763301

K283A

the mutation reduces activity to the background level

K377A

the mutation reduces activity to the background level

Q180A

he mutation decreases the enzyme activity by 64% compared to the wild type enzyme

additional information

6 entries

6.8

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3 h, no significant loss of activity at 20°C and at 30°C

8.7

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1 h, 50% loss of activity at 20°C and 75% at 30°C. At 30°C, the loss is reduced to 40% in the presence of 0.1 mg/ml microtubules

20

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pH 8.7, 1 h, 50% loss of activity. Not significant loss of activity within 3 h of incubation at pH 6.8

30

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pH 8.7, 1 h, 75% loss of activity. No significant loss of activity within 3 h of incubation at pH 6.8

at 30°C, the loss of activity is reduced to 40% in the presence of 0.1 mg/ml microtubules, while neither bovine serum albumin nor the other substrates, ATP or glutamate, have any protecting effect

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C11ORF49/CSTPP1 regulates the assembly and stability of the enzyme complex and thereby modulates polyglutamylation of microtubule, which antagonizes MAP4 binding

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762893

-80°C, 10% glycerol, stable for several months

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-80°C, in the presence of 10% (v/v) glycerol, several months, no loss of activity

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20°C, in 10 mM Tris (pH 8.7), 1 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, and 0.01% TX-100 at pH 8.7, 1 h, 50% loss of activity

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30°C, in 10 mM Tris (pH 8.7), 1 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, and 0.01% TX-100 at pH 8.7, 1 h, 75% loss of activity

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3 entries

ATP-affinity chromatography, glycerol-gradient centrifugation and Mono Q ion-exchange chromatography

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GST affinity chromatography, heparin column chromatography, and Superdex 75 gel filtration

partial

Q23SI8, I7MCG4

750454

partial purification of recombinant GFP-tagged Ttll1p and GFP-tagged Ttll9p

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partially purified by phosphocellulose column chromatography

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763304

phosphocellulose column chromatography, MgCl2 and sodium citrate precipitation, sucrose gradient sedimentation, and TSK G3000 gel filtration

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Q23SI8, I7MCG4

750454

expressed in Escherichia coli Arctic Express (DE3) cells

expressed in Escherichia coli BL21(DE3) cells

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763305

expressed in HeLa cells

Q91V51, Q8CHB8, A4Q9E8, A4Q9F4, A4Q9F6

751550

expressed in HeLa cells or U2OS cells

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763301

expression of GFP-tagged Ttll1p mainly in the basal bodies and of GFP-tagged Ttll9p mainly in the cilia of CU428 and B2086 cells

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enzyme activity is maximal in G2-phase and decreases during M-phase of the cell cycle