EC Number | Cloned (Comment) | Organism |
---|---|---|
2.7.7.101 | gene dnaG, recombinant expression of His-tagged wild-type and mutant enzymes, and of unlabeled and selenomethionine-labeled MtDnaG-CTDs in Escherichia coli strain BL21(DE3) | Mycobacterium tuberculosis |
EC Number | Crystallization (Comment) | Organism |
---|---|---|
2.7.7.101 | purified recombinant C-terminal domain of DnaG, mixing of 0.002 ml of 6 mg/ml native MtDnaG-CTD and 5 mg/ml selenomethionine-labeled MtDnaG-CTD with 0.002 ml of reservoir solution containing 20% w/v PEG 3350, 0.2 M magnesium acetate tetrahydrate, and equilibration against 0.5 ml of reservoir solution, for the SeMet-labeled protein a reeservoir solution of reservoir solution of 18% w/v PEG 3350, 0.15 M magnesium acetate, and MOPS, pH 7.6, is used, 4°C, 1-2 weeks, X-ray diffraction structure determination and analysis at 1.57-1.58 A resolution, modeling | Mycobacterium tuberculosis |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
2.7.7.101 | F615A | site-directed mutagenesis, mutant MtDnaG-CTD F615A | Mycobacterium tuberculosis |
2.7.7.101 | I605A | site-directed mutagenesis, mutant MtDnaG-CTD I605A, binding study shows a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD | Mycobacterium tuberculosis |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
2.7.7.101 | Mg2+ | required | Mycobacterium tuberculosis |
EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|---|
2.7.7.101 | 35000 | - |
the C-terminal domain MtDnaG-CTD is a dimer in solution, gel filtration | Mycobacterium tuberculosis |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.7.7.101 | ssDNA + n NTP | Mycobacterium tuberculosis | - |
ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate | - |
? | |
2.7.7.101 | ssDNA + n NTP | Mycobacterium tuberculosis H37Rv | - |
ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate | - |
? | |
2.7.7.101 | ssDNA + n NTP | Mycobacterium tuberculosis ATCC 25618 | - |
ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
2.7.7.101 | Mycobacterium tuberculosis | P9WNW1 | - |
- |
2.7.7.101 | Mycobacterium tuberculosis ATCC 25618 | P9WNW1 | - |
- |
2.7.7.101 | Mycobacterium tuberculosis H37Rv | P9WNW1 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
2.7.7.101 | recombinant His-tagged -type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ultrafiltration, and gel filtration | Mycobacterium tuberculosis |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.7.7.101 | ssDNA + n NTP | - |
Mycobacterium tuberculosis | ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate | - |
? | |
2.7.7.101 | ssDNA + n NTP | - |
Mycobacterium tuberculosis H37Rv | ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate | - |
? | |
2.7.7.101 | ssDNA + n NTP | - |
Mycobacterium tuberculosis ATCC 25618 | ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate | - |
? |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
2.7.7.101 | More | MtDnaG-CTD interacts at the dimer-dimer interface of MtDnaB-NTD using mostly hydrophobic residues of the helical hairpin regions (HHRs). The dimer-dimer interface of MtDnaB-NTD is considered as a DnaG-binding site | Mycobacterium tuberculosis |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
2.7.7.101 | DnaG | - |
Mycobacterium tuberculosis |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
2.7.7.101 | 37 | - |
assay at | Mycobacterium tuberculosis |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
2.7.7.101 | 7.5 | - |
assay at | Mycobacterium tuberculosis |
EC Number | General Information | Comment | Organism |
---|---|---|---|
2.7.7.101 | evolution | functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria. MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae. The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system | Mycobacterium tuberculosis |
2.7.7.101 | additional information | structure modeling using the MtDnaG-CTD and MtDnaB-NTD crystal structures for analysis of crucial helicase-primase interaction in Mycobacterium tuberculosis. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG are identified as potential key residues interacting with MtDnaB. The loop, connecting the two helices of the HHR, is concluded to be largely responsible for the stability of the DnaB-DnaG complex. Molecular dynamic simulations were performed on the models of the MtDnaB-NTD complex with DnaG-CTD, DnaG-CTD I605A, and DnaG-CTD F615A. Structure comparisons of the enzyme from Mycobacterium tuberculosis with enzymes from other species. Residue Ile605 in the HHR is crucial for helicase-primase interaction. The dimer-dimer interface of MtDnaB-NTD is considered as a DnaG-binding site. Mycobacterium DnaB-NTD interacts with DnaG-CTD of other organisms with low binding affinity | Mycobacterium tuberculosis |
2.7.7.101 | physiological function | the helicase-primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain (CTD) of primase (DnaG) and N-terminal domain (NTD) of helicase (DnaB) | Mycobacterium tuberculosis |