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Literature summary for 5.6.1.4 extracted from

  • Szczesna, E.; Kasprzak, A.A.
    The C-terminus of kinesin-14 Ncd is a crucial component of the force generating mechanism (2012), FEBS Lett., 586, 854-858.
    View publication on PubMed
Search for more literature for 5.6.1.4

Activating Compound

Activating Compound Comment Organism Structure
additional information microtubule-stimulated ATPase Drosophila melanogaster

Cloned(Commentary)

Cloned (Comment) Organism
His tagged truncated proteins (e.g. wild type, residue 250-700) expressed in Escherichia coli Drosophila melanogaster
recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli Drosophila melanogaster

Protein Variants

Protein Variants Comment Organism
M672N site-directed mutagenesis, the mutation has a negligible effect on microtubule binding in the absence of nucleotides although substantially changed the Kd(MT) in the presence of AMPPNP Drosophila melanogaster
additional information truncated versions: residue 250-700 (wild type), residue 346-700 (motor domain 700), residue 250-670 (Ncd670), residue 346-700 (motor domain 670), residue 250-679 (Ncd679), residue 250-670 + GGSGGSGGS + 680-700 (Ncd700SG, residue 671-679 replaced by sequence GGSGGSGGS), residue 250-670 with M672N exchange (NcdM672N) Drosophila melanogaster
additional information production of four Ncd mutants in which the C-terminus is altered or deleted, analysis of mutant affinity to the microtubule, steady-state ATPase and gliding velocity in multiple motor assays: the mutations have a dramatic effect on all three parameters measured, suggesting that the C-terminal residues of Ncd play an important role in modulating the interaction of the motor with the microtubule. In the first of the mutants, Ncd670, the entire C-terminal segment is deleted. In mutant Ncd679, the basic fragment spanning aa 671–679 is left intact whereas the last 21 aa are deleted. In mutant Ncd700SG the basic fragment is replaced by the sequence GGSGGSGGS, which is uncharged and flexible. In the fourth construct NcdM672N, the hydrophobic Met672 is substituted by a small and polar Asn Drosophila melanogaster

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.00014
-
 ATP mutants Ncd670 and Ncd700SG, pH 7.2, 25°C, microtubule-stimulated ATPase activity Drosophila melanogaster
0.00022
-
 ATP wild-type Ncd, pH 7.2, 25°C, microtubule-stimulated ATPase activity Drosophila melanogaster
0.00026
-
 ATP mutant Ncd679, pH 7.2, 25°C, microtubule-stimulated ATPase activity Drosophila melanogaster
0.00061
-
 ATP mutant NcdM672N, pH 7.2, 25°C, microtubule-stimulated ATPase activity Drosophila melanogaster

Localization

Localization Comment Organism GeneOntology No. Textmining
microtubule
-
 Drosophila melanogaster 5874
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Drosophila melanogaster

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + H2O + a kinesin associated with a microtubule at position n Drosophila melanogaster
-
 ADP + phosphate + a kinesin associated with a microtubule at position n-1 (toward the minus end)
-
 ?

Organism

Organism UniProt Comment Textmining
Drosophila melanogaster
-
-
-

Purification (Commentary)

Purification (Comment) Organism
immobilized metal ion affinity chromatography (Ni2+) Drosophila melanogaster
recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli Drosophila melanogaster

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + H2O + a kinesin associated with a microtubule at position n
-
 Drosophila melanogaster ADP + phosphate + a kinesin associated with a microtubule at position n-1 (toward the minus end)
-
 ?
additional information measurement of Ncd affinity to microtubules, microtubule-stimulated ATPase, and motility of wild-type and mutant enzymes Drosophila melanogaster ?
-
 ?

Subunits

Subunits Comment Organism
dimer
-
 Drosophila melanogaster
homodimer
-
 Drosophila melanogaster

Synonyms

Synonyms Comment Organism
kinesin-14
-
 Drosophila melanogaster
kinesin-14 Ncd
-
 Drosophila melanogaster
ncd
-
 Drosophila melanogaster
non-claret disjunctional
-
 Drosophila melanogaster

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Drosophila melanogaster

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.62
-
 ATP Ncd700SG (residue 250-700, residue 671-679 replaced by sequence GGSGGSGGS), microtuble stimulated, Vmax/2 at 0.00014 mM microtubles, pH 7.2, 25°C Drosophila melanogaster
0.62
-
 ATP mutant Ncd700SG, pH 7.2, 25°C, microtubule-stimulated ATPase activity Drosophila melanogaster
0.83
-
 ATP Ncd670 (residue 250-670), microtuble stimulated, Vmax/2 at 0.00014 mM microtubles, pH 7.2, 25°C Drosophila melanogaster
0.83
-
 ATP mutant Ncd670, pH 7.2, 25°C, microtubule-stimulated ATPase activity Drosophila melanogaster
2.49
-
 ATP NcdM672N (residue 250-679, M672N mutation), microtuble stimulated, Vmax/2 at 0.00061 mM microtubles, pH 7.2, 25°C Drosophila melanogaster
2.49
-
 ATP mutant NcdM672N, pH 7.2, 25°C, microtubule-stimulated ATPase activity Drosophila melanogaster
2.58
-
 ATP NcdWT (residue 250-700), microtuble stimulated, Vmax/2 at 0.00022 mM microtubles, pH 7.2, 25°C Drosophila melanogaster
2.58
-
 ATP wild-type Ncd, pH 7.2, 25°C, microtubule-stimulated ATPase activity Drosophila melanogaster
3.58
-
 ATP Ncd679 (residue 250-679), microtuble stimulated, Vmax/2 at 0.00026 mM microtubles, pH 7.2, 25°C Drosophila melanogaster
3.58
-
 ATP mutant Ncd679, pH 7.2, 25°C, microtubule-stimulated ATPase activity Drosophila melanogaster

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.2
-
 assay at Drosophila melanogaster

General Information

General Information Comment Organism
evolution Ncd is a member of kinesin superfamily, kinesin-14 subfamily motors that uses the power stroke, a lever-like pivoting action of a long and stiff element, to exert force and generate movement Drosophila melanogaster
additional information the C-terminal segment of the kinesin-14 subfamily is formed by residues that extend beyond the a6 helix Drosophila melanogaster
physiological function Ncd is a member of kinesin-14 subfamily motors that uses the power stroke, a lever-like pivoting action of a long and stiff element, to exert force and generate movement, the Ncd C-terminus is crucial in this process modulating the interaction of the motor with the microtubule. Ncd is a retrograde motor that participates in spindle assembly by generating microtubule-microtubule sliding in an orientation dependent manner Drosophila melanogaster