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Literature summary for 6.5.1.2 extracted from
- NAD+-dependent DNA ligase encoded by a eukaryotic virus (2001), J. Biol. Chem., 276, 36100-36109.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| native and mutant enzymes in Escherichia coli BL21 (DE3) | Amsacta moorei entomopoxvirus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D117A | no nick-ligation activity | Amsacta moorei entomopoxvirus |
| D27A | no effect on activity | Amsacta moorei entomopoxvirus |
| D48A | reduced adenylate-transfer activity, no effect on DNA-ligation with preadenylated nick | Amsacta moorei entomopoxvirus |
| D52A | reduced adenylate-transfer activity, no effect on DNA-ligation with preadenylated nick | Amsacta moorei entomopoxvirus |
| DEL1-70 | inactive in overall ligation reaction, fully functional with preadenylated nick | Amsacta moorei entomopoxvirus |
| DEL216-532 | inactive in overall ligation reaction, formation of adenylate-ligase intermediate not affected | Amsacta moorei entomopoxvirus |
| K115A | no nick-ligation activity | Amsacta moorei entomopoxvirus |
| Y39A | reduced adenylate-transfer activity, no effect on DNA-ligation with preadenylated nick | Amsacta moorei entomopoxvirus |
| Y40A | reduced adenylate-transfer activity, no effect on DNA-ligation with preadenylated nick | Amsacta moorei entomopoxvirus |
| Y51A | adenylate-transfer activity abolished, no effect on DNA-ligation with preadenylated nick | Amsacta moorei entomopoxvirus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.009 | - |
NAD+ | - |
Amsacta moorei entomopoxvirus |  |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Co2+ | can partially substitute for Mg2+ | Amsacta moorei entomopoxvirus | |
| Mg2+ | highest activity at 5 mM | Amsacta moorei entomopoxvirus | |
| Mn2+ | can partially substitute for Mg2+ | Amsacta moorei entomopoxvirus | |
| additional information | Ca2+, Zn2+ and Cu2+ have no effect on activity | Amsacta moorei entomopoxvirus | |
| additional information | no zinc binding motif as known from most bacterial DNA-ligases | Amsacta moorei entomopoxvirus |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 62000 | - |
SDS-PAGE, recombinant enzyme | Amsacta moorei entomopoxvirus |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m | Amsacta moorei entomopoxvirus | DNA-repair, phosphodiester bond formation between adjacent 5'-phosphate and 3'-hydroxyl groups in double-stranded DNA | AMP + nicotinamide nucleotide + (deoxyribonucleotide)n+m | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Amsacta moorei entomopoxvirus | - |
entomopoxvirus | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant enzymes using His-tag | Amsacta moorei entomopoxvirus |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| ATP + (deoxyribonucleotide)n-3'-hydroxyl + 5'-phospho-(deoxyribonucleotide)m = (deoxyribonucleotide)n+m + AMP + beta-nicotinamide D-nucleotide | 3 steps of reaction: 1. adenylation of the ligase in the presence of NAD+, 2. transferring the adenylate moiety to the 5'-phosphate of the nicked DNA substrate, 3. sealing the nick through the formation of a phosphodiester bond | Amsacta moorei entomopoxvirus |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -80°C | Amsacta moorei entomopoxvirus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m | - |
Amsacta moorei entomopoxvirus | AMP + nicotinamide nucleotide + (deoxyribonucleotide)n+m | - |
? | |
| NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m | DNA-repair, phosphodiester bond formation between adjacent 5'-phosphate and 3'-hydroxyl groups in double-stranded DNA | Amsacta moorei entomopoxvirus | AMP + nicotinamide nucleotide + (deoxyribonucleotide)n+m | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| monomer | zonal velocity sedimentation centrifugation, protein is found to be asymmetrically shaped | Amsacta moorei entomopoxvirus |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 6.5 | 9 | - |
Amsacta moorei entomopoxvirus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| NAD+ | required, highest activity 0.005-0.1 mM | Amsacta moorei entomopoxvirus | |
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