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Literature summary for 3.6.1.15 extracted from
- Crystal structure and biochemical characterization of a Mycobacterium smegmatis AAA-type nucleoside triphosphatase phosphohydrolase (Msm0858) (2016), J. Bacteriol., 198, 1521-1533 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| 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)
| Crystallization (Comment) | Organism |
|---|---|
| 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
| Protein Variants | Comment | Organism |
|---|---|---|
| D327A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Mycolicibacterium smegmatis |
| D327A/E585A | site-directed mutagenesis, inactive mutant | Mycolicibacterium smegmatis |
| E585A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Mycolicibacterium smegmatis |
| K276A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Mycolicibacterium smegmatis |
| K276A/K532A | site-directed mutagenesis, inactive mutant | Mycolicibacterium smegmatis |
| K532A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Mycolicibacterium smegmatis |
| 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 |
| 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
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Ca2+ | high activation | Mycolicibacterium smegmatis |  |
| Mg2+ | dependent on, highest activation | Mycolicibacterium smegmatis | |
| Mn2+ | moderate activation | Mycolicibacterium smegmatis | |
| additional information | poor activation by Cu2+, Cd2+, Co2+, and Zn2+, no activation by Ni2+ | Mycolicibacterium smegmatis |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 78000 | - |
glycerol gradient sedimentation centrifugation | Mycolicibacterium smegmatis |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O | Mycolicibacterium smegmatis | - |
ADP + phosphate | - |
? | |
| ATP + H2O | Mycolicibacterium smegmatis ATCC 700084 | - |
ADP + phosphate | - |
? | |
| ATP + H2O | Mycolicibacterium smegmatis mc(2)155 | - |
ADP + phosphate | - |
? | |
| CTP + H2O | Mycolicibacterium smegmatis | - |
CDP + phosphate | - |
? | |
| CTP + H2O | Mycolicibacterium smegmatis ATCC 700084 | - |
CDP + phosphate | - |
? | |
| CTP + H2O | Mycolicibacterium smegmatis mc(2)155 | - |
CDP + phosphate | - |
? | |
| dATP + H2O | Mycolicibacterium smegmatis | - |
dADP + phosphate | - |
? | |
| dCTP + H2O | Mycolicibacterium smegmatis | - |
dCDP + phosphate | - |
? | |
| dGTP + H2O | Mycolicibacterium smegmatis | best substrate | dGDP + phosphate | - |
? | |
| dTTP + H2O | Mycolicibacterium smegmatis | - |
dTDP + phosphate | - |
? | |
| GTP + H2O | Mycolicibacterium smegmatis | - |
GDP + phosphate | - |
? | |
| GTP + H2O | Mycolicibacterium smegmatis ATCC 700084 | - |
GDP + phosphate | - |
? | |
| GTP + H2O | Mycolicibacterium smegmatis mc(2)155 | - |
GDP + phosphate | - |
? | |
| UTP + H2O | Mycolicibacterium smegmatis | - |
UDP + phosphate | - |
? | |
| UTP + H2O | Mycolicibacterium smegmatis ATCC 700084 | - |
UDP + phosphate | - |
? | |
| UTP + H2O | Mycolicibacterium smegmatis mc(2)155 | - |
UDP + phosphate | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Mycolicibacterium smegmatis | A0QQS4 | i.e. Mycobacterium smegmatis | - |
| Mycolicibacterium smegmatis ATCC 700084 | A0QQS4 | i.e. Mycobacterium smegmatis | - |
| Mycolicibacterium smegmatis mc(2)155 | A0QQS4 | i.e. Mycobacterium smegmatis | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| 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)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O | - |
Mycolicibacterium smegmatis | ADP + phosphate | - |
? | |
| ATP + H2O | - |
Mycolicibacterium smegmatis ATCC 700084 | ADP + phosphate | - |
? | |
| ATP + H2O | - |
Mycolicibacterium smegmatis mc(2)155 | ADP + phosphate | - |
? | |
| CTP + H2O | - |
Mycolicibacterium smegmatis | CDP + phosphate | - |
? | |
| CTP + H2O | - |
Mycolicibacterium smegmatis ATCC 700084 | CDP + phosphate | - |
? | |
| CTP + H2O | - |
Mycolicibacterium smegmatis mc(2)155 | CDP + phosphate | - |
? | |
| dATP + H2O | - |
Mycolicibacterium smegmatis | dADP + phosphate | - |
? | |
| dCTP + H2O | - |
Mycolicibacterium smegmatis | dCDP + phosphate | - |
? | |
| dGTP + H2O | - |
Mycolicibacterium smegmatis | dGDP + phosphate | - |
? | |
| dGTP + H2O | best substrate | Mycolicibacterium smegmatis | dGDP + phosphate | - |
? | |
| dTTP + H2O | - |
Mycolicibacterium smegmatis | dTDP + phosphate | - |
? | |
| dUTP + H2O | - |
Mycolicibacterium smegmatis | dUDP + phosphate | - |
? | |
| GTP + H2O | - |
Mycolicibacterium smegmatis | GDP + phosphate | - |
? | |
| GTP + H2O | - |
Mycolicibacterium smegmatis ATCC 700084 | GDP + phosphate | - |
? | |
| GTP + H2O | - |
Mycolicibacterium smegmatis mc(2)155 | GDP + phosphate | - |
? | |
| 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 | ? | - |
- |
|
| 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 | ? | - |
- |
|
| 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 | ? | - |
- |
|
| UTP + H2O | - |
Mycolicibacterium smegmatis | UDP + phosphate | - |
? | |
| UTP + H2O | - |
Mycolicibacterium smegmatis ATCC 700084 | UDP + phosphate | - |
? | |
| UTP + H2O | - |
Mycolicibacterium smegmatis mc(2)155 | UDP + phosphate | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| monomer | 1 * 78000, SDS-PAGE | Mycolicibacterium smegmatis |
| 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
| Synonyms | Comment | Organism |
|---|---|---|
| AAA-type nucleoside triphosphatase phosphohydrolase | - |
Mycolicibacterium smegmatis |
| cell division control protein Cdc48 | UniProt | Mycolicibacterium smegmatis |
| Msm0858 | - |
Mycolicibacterium smegmatis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Mycolicibacterium smegmatis |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Mycolicibacterium smegmatis |
General Information
| General Information | Comment | Organism |
|---|---|---|
| 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 |
| 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 |
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