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Literature summary extracted from

  • Unciuleac, M.C.; Smith, P.C.; Shuman, S.
    Crystal structure and biochemical characterization of a Mycobacterium smegmatis AAA-type nucleoside triphosphatase phosphohydrolase (Msm0858) (2016), J. Bacteriol., 198, 1521-1533 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.6.1.15 gene MSMEG_0858, recombinant expression of N-terminally His10-tagged enzyme in Escherichia coli strain BL21(DE3) and of selenomethionine (SeMet)-substituted Msm0858 in Escherichia coli strain B834 Mycolicibacterium smegmatis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.6.1.15 purified recombinant wild-type enzyme and SeMet-labeled enzyme, hanging drop vapor diffusion method, mixing of 6.5 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 10 mM DTT, with an equal volume of reservoir solution containing 0.3 M ammonium tartrate dibasic and 25% PEG 3350, room temperature, 1 week, X-ray diffraction structure determination and analysis at 2.5 and 3.2 A resolution, respectively Mycolicibacterium smegmatis

Protein Variants

EC Number Protein Variants Comment Organism
3.6.1.15 D327A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Mycolicibacterium smegmatis
3.6.1.15 D327A/E585A site-directed mutagenesis, inactive mutant Mycolicibacterium smegmatis
3.6.1.15 E585A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Mycolicibacterium smegmatis
3.6.1.15 K276A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Mycolicibacterium smegmatis
3.6.1.15 K276A/K532A site-directed mutagenesis, inactive mutant Mycolicibacterium smegmatis
3.6.1.15 K532A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Mycolicibacterium smegmatis
3.6.1.15 additional information construction of a truncated variant Msm0858 (amino acids 212-745) that lacks the N domain, the mutant is a catalytically active homodimer, quarternary strutcure analysis, overview Mycolicibacterium smegmatis
3.6.1.15 R641A site-directed mutagenesis, Arg641 is the predicted arginine finger of the D2 AAA domain based on the structural alignment of Msm0858 to p97, almost inactive mutant Mycolicibacterium smegmatis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.6.1.15 Ca2+ high activation Mycolicibacterium smegmatis
3.6.1.15 Mg2+ dependent on, highest activation Mycolicibacterium smegmatis
3.6.1.15 Mn2+ moderate activation Mycolicibacterium smegmatis
3.6.1.15 additional information poor activation by Cu2+, Cd2+, Co2+, and Zn2+, no activation by Ni2+ Mycolicibacterium smegmatis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.6.1.15 78000
-
 glycerol gradient sedimentation centrifugation Mycolicibacterium smegmatis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.6.1.15 ATP + H2O Mycolicibacterium smegmatis
-
 ADP + phosphate
-
 ?
3.6.1.15 ATP + H2O Mycolicibacterium smegmatis ATCC 700084
-
 ADP + phosphate
-
 ?
3.6.1.15 ATP + H2O Mycolicibacterium smegmatis mc(2)155
-
 ADP + phosphate
-
 ?
3.6.1.15 CTP + H2O Mycolicibacterium smegmatis
-
 CDP + phosphate
-
 ?
3.6.1.15 CTP + H2O Mycolicibacterium smegmatis ATCC 700084
-
 CDP + phosphate
-
 ?
3.6.1.15 CTP + H2O Mycolicibacterium smegmatis mc(2)155
-
 CDP + phosphate
-
 ?
3.6.1.15 dATP + H2O Mycolicibacterium smegmatis
-
 dADP + phosphate
-
 ?
3.6.1.15 dCTP + H2O Mycolicibacterium smegmatis
-
 dCDP + phosphate
-
 ?
3.6.1.15 dGTP + H2O Mycolicibacterium smegmatis best substrate dGDP + phosphate
-
 ?
3.6.1.15 dTTP + H2O Mycolicibacterium smegmatis
-
 dTDP + phosphate
-
 ?
3.6.1.15 GTP + H2O Mycolicibacterium smegmatis
-
 GDP + phosphate
-
 ?
3.6.1.15 GTP + H2O Mycolicibacterium smegmatis ATCC 700084
-
 GDP + phosphate
-
 ?
3.6.1.15 GTP + H2O Mycolicibacterium smegmatis mc(2)155
-
 GDP + phosphate
-
 ?
3.6.1.15 UTP + H2O Mycolicibacterium smegmatis
-
 UDP + phosphate
-
 ?
3.6.1.15 UTP + H2O Mycolicibacterium smegmatis ATCC 700084
-
 UDP + phosphate
-
 ?
3.6.1.15 UTP + H2O Mycolicibacterium smegmatis mc(2)155
-
 UDP + phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.6.1.15 Mycolicibacterium smegmatis A0QQS4 i.e. Mycobacterium smegmatis
-
3.6.1.15 Mycolicibacterium smegmatis ATCC 700084 A0QQS4 i.e. Mycobacterium smegmatis
-
3.6.1.15 Mycolicibacterium smegmatis mc(2)155 A0QQS4 i.e. Mycobacterium smegmatis
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.6.1.15 recombinant wild-type or SeMet-labeled His10-tagged enzyme from Escherichia coli strains BL21(DE3) or B834 by nickel affinity chromatography, tag cleavage by Smt3-specific protease Ulp1, a second step of nickel affinity chromatography, and ultrafiltration, followed by dialysis Mycolicibacterium smegmatis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.6.1.15 ATP + H2O
-
 Mycolicibacterium smegmatis ADP + phosphate
-
 ?
3.6.1.15 ATP + H2O
-
 Mycolicibacterium smegmatis ATCC 700084 ADP + phosphate
-
 ?
3.6.1.15 ATP + H2O
-
 Mycolicibacterium smegmatis mc(2)155 ADP + phosphate
-
 ?
3.6.1.15 CTP + H2O
-
 Mycolicibacterium smegmatis CDP + phosphate
-
 ?
3.6.1.15 CTP + H2O
-
 Mycolicibacterium smegmatis ATCC 700084 CDP + phosphate
-
 ?
3.6.1.15 CTP + H2O
-
 Mycolicibacterium smegmatis mc(2)155 CDP + phosphate
-
 ?
3.6.1.15 dATP + H2O
-
 Mycolicibacterium smegmatis dADP + phosphate
-
 ?
3.6.1.15 dCTP + H2O
-
 Mycolicibacterium smegmatis dCDP + phosphate
-
 ?
3.6.1.15 dGTP + H2O
-
 Mycolicibacterium smegmatis dGDP + phosphate
-
 ?
3.6.1.15 dGTP + H2O best substrate Mycolicibacterium smegmatis dGDP + phosphate
-
 ?
3.6.1.15 dTTP + H2O
-
 Mycolicibacterium smegmatis dTDP + phosphate
-
 ?
3.6.1.15 dUTP + H2O
-
 Mycolicibacterium smegmatis dUDP + phosphate
-
 ?
3.6.1.15 GTP + H2O
-
 Mycolicibacterium smegmatis GDP + phosphate
-
 ?
3.6.1.15 GTP + H2O
-
 Mycolicibacterium smegmatis ATCC 700084 GDP + phosphate
-
 ?
3.6.1.15 GTP + H2O
-
 Mycolicibacterium smegmatis mc(2)155 GDP + phosphate
-
 ?
3.6.1.15 additional information enzyme Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates Mycolicibacterium smegmatis ?
-
-
3.6.1.15 additional information enzyme Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates Mycolicibacterium smegmatis ATCC 700084 ?
-
-
3.6.1.15 additional information enzyme Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates Mycolicibacterium smegmatis mc(2)155 ?
-
-
3.6.1.15 UTP + H2O
-
 Mycolicibacterium smegmatis UDP + phosphate
-
 ?
3.6.1.15 UTP + H2O
-
 Mycolicibacterium smegmatis ATCC 700084 UDP + phosphate
-
 ?
3.6.1.15 UTP + H2O
-
 Mycolicibacterium smegmatis mc(2)155 UDP + phosphate
-
 ?

Subunits

EC Number Subunits Comment Organism
3.6.1.15 monomer 1 * 78000, SDS-PAGE Mycolicibacterium smegmatis
3.6.1.15 More the Msm0858 structure comprises (i) an N-terminal domain (amino acids 17-201) composed of two beta-barrel modules and (ii) two AAA domains, D1 (amino acids 212-473) and D2 (amino acids 476-744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. The D1 and D2 AAA domains are both capable of ATP hydrolysis, Enzyme structure analysis, detailed overview. Msm0858 might oligomerize during the ATPase reaction cycle Mycolicibacterium smegmatis

Synonyms

EC Number Synonyms Comment Organism
3.6.1.15 AAA-type nucleoside triphosphatase phosphohydrolase
-
 Mycolicibacterium smegmatis
3.6.1.15 cell division control protein Cdc48 UniProt Mycolicibacterium smegmatis
3.6.1.15 Msm0858
-
 Mycolicibacterium smegmatis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.6.1.15 37
-
 assay at Mycolicibacterium smegmatis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.6.1.15 8
-
 assay at Mycolicibacterium smegmatis

General Information

EC Number General Information Comment Organism
3.6.1.15 malfunction simultaneous mutations of the D1 and D2 active-site motifs are required to abolish ATPase activity. ATPase activity is effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (amino acids 212-745) that lacks the N domain is characterized as a catalytically active homodimer Mycolicibacterium smegmatis
3.6.1.15 additional information mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis, structure comparisons of the enzymes with mammalian protein p97, homology of the tandem AAA domains of Msm0858 and p97, overview Mycolicibacterium smegmatis