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Literature summary for 4.2.99.18 extracted from
- Critical determinants for substrate recognition and catalysis in the M. tuberculosis class II AP-endonuclease/3'-5' exonuclease III (2015), Biochim. Biophys. Acta, 1854, 505-516 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of wild-type and mutant enzymes | Mycobacterium tuberculosis |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D251A | site-directed mutagenesis, the mutant shows a 3fold decline in the endonucleolytic cleavage activity | Mycobacterium tuberculosis |
| E57A | site-directed mutagenesis, the mutant shows a 2fold decline in the endonucleolytic cleavage activity | Mycobacterium tuberculosis |
| E57A/D251A | site-directed mutagenesis, the double mutant shows no 3'-5' exonuclease activity | Mycobacterium tuberculosis |
| F242S | site-directed mutagenesis, the mutant shows increased 3'-5' exonuclease catalytic efficiency compared to wild-type | Mycobacterium tuberculosis |
| W235S | site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type | Mycobacterium tuberculosis |
| Y137S | site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type | Mycobacterium tuberculosis |
| Y234S | site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type | Mycobacterium tuberculosis |
| Y234S/W235S | site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type | Mycobacterium tuberculosis |
| Y237S | site-directed mutagenesis, the mutant shows no 3'-5' exonuclease activity | Mycobacterium tuberculosis |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | steady-state Michaelis-Menten kinetic analysis | Mycobacterium tuberculosis |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required, MtbXthA is inactive in AP site incision assays in the absence of Mg2+, while increasing activity is observed with increasing Mg2+ concentration between 1-10 mM. The protein exhibits maximal incision activity, 60%, when no NaCl is included in the buffer | Mycobacterium tuberculosis |  |
| additional information | the highly conserved catalytic site in AP endonucleases consists of residues involved in the binding of metal ions. MtbXthA exhibits moderate 3'-5' exonuclease activity at low ionic environment | Mycobacterium tuberculosis | |
| NaCl | MtbXthA exhibits an increase in AP site incision activity in a salt-dependent manner. Optimum conditions are 2 mM MgCl2 and 150 mM NaCl with 75% incision activity. Addition of more than 200 mM salt, strongly inhibits the endonuclease activity | Mycobacterium tuberculosis | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Mycobacterium tuberculosis | P96273 | - |
- |
| Mycobacterium tuberculosis ATCC 25618 | P96273 | - |
- |
| Mycobacterium tuberculosis H37Rv | P96273 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant wild-type and mutant enzymes | Mycobacterium tuberculosis |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| additional information | XthA is expressed constitutively | Mycobacterium tuberculosis | - |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | nicked DNA and gapped DNA are fair substrates of enzyme MtbXthA, the gap-size does not affect the excision activity, a substrate with a recessed 3'-end is preferred. DNA substrate N1 containing an abasic site analogue tetrahydrofuran (THF), is used for AP site incision activity assays. Substrate N2 that contains propanediol, tetrahydrofuran, ethane or 2' deoxyribose as the abasic residues is used for studying the effect of abasic site structure on the AP site incision activity of MtbXthA. For the preparation of the duplex DNA containing a 2' deoxyribose as abasic residue, first an oligonucleotide containing a 2' deoxyribose is prepared by enzymatic treatment of an oligonucleotide harbouring 2' deoxyuridine with uracil N-glycosylase. DNA elements have no role in the recognition of AP site containing DNA by MtbXthA. Y237 functions as a critical AP site recogniser in MtbXthA. Analysis of the 3' phosphatase and 3' phosphodiesterase activities of MtbXthA with a DNA substrate | Mycobacterium tuberculosis | ? | - |
? | |
| additional information | nicked DNA and gapped DNA are fair substrates of enzyme MtbXthA, the gap-size does not affect the excision activity, a substrate with a recessed 3'-end is preferred. DNA substrate N1 containing an abasic site analogue tetrahydrofuran (THF), is used for AP site incision activity assays. Substrate N2 that contains propanediol, tetrahydrofuran, ethane or 2' deoxyribose as the abasic residues is used for studying the effect of abasic site structure on the AP site incision activity of MtbXthA. For the preparation of the duplex DNA containing a 2' deoxyribose as abasic residue, first an oligonucleotide containing a 2' deoxyribose is prepared by enzymatic treatment of an oligonucleotide harbouring 2' deoxyuridine with uracil N-glycosylase. DNA elements have no role in the recognition of AP site containing DNA by MtbXthA. Y237 functions as a critical AP site recogniser in MtbXthA. Analysis of the 3' phosphatase and 3' phosphodiesterase activities of MtbXthA with a DNA substrate | Mycobacterium tuberculosis H37Rv | ? | - |
? | |
| additional information | nicked DNA and gapped DNA are fair substrates of enzyme MtbXthA, the gap-size does not affect the excision activity, a substrate with a recessed 3'-end is preferred. DNA substrate N1 containing an abasic site analogue tetrahydrofuran (THF), is used for AP site incision activity assays. Substrate N2 that contains propanediol, tetrahydrofuran, ethane or 2' deoxyribose as the abasic residues is used for studying the effect of abasic site structure on the AP site incision activity of MtbXthA. For the preparation of the duplex DNA containing a 2' deoxyribose as abasic residue, first an oligonucleotide containing a 2' deoxyribose is prepared by enzymatic treatment of an oligonucleotide harbouring 2' deoxyuridine with uracil N-glycosylase. DNA elements have no role in the recognition of AP site containing DNA by MtbXthA. Y237 functions as a critical AP site recogniser in MtbXthA. Analysis of the 3' phosphatase and 3' phosphodiesterase activities of MtbXthA with a DNA substrate | Mycobacterium tuberculosis ATCC 25618 | ? | - |
? | |
| N1 duplex DNA substrate + H2O | 5'-FAM peptide-labelled AP DNA duplex substrate | Mycobacterium tuberculosis | ? | - |
? | |
| N1 duplex DNA substrate + H2O | 5'-FAM peptide-labelled AP DNA duplex substrate | Mycobacterium tuberculosis H37Rv | ? | - |
? | |
| N1 duplex DNA substrate + H2O | 5'-FAM peptide-labelled AP DNA duplex substrate | Mycobacterium tuberculosis ATCC 25618 | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| AP endonuclease VI | UniProt | Mycobacterium tuberculosis |
| AP-endonuclease | - |
Mycobacterium tuberculosis |
| AP-endonuclease/3'-5'exodeoxyribonuclease | - |
Mycobacterium tuberculosis |
| More | cf. EC 3.1.11.2, 3'-5' exonuclease III | Mycobacterium tuberculosis |
| MtbXthA | - |
Mycobacterium tuberculosis |
| xthA | - |
Mycobacterium tuberculosis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Mycobacterium tuberculosis |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.8 | - |
assay at | Mycobacterium tuberculosis |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | AP endonucleases have been classified into XthA and Nfo families based on sequence homology and structural conservation studies involving Escherichia coli exonuclease III (ExoIII) or endonuclease IV (EndoIV) respectively. The catalytic site in AP endonucleases is highly conserved from bacteria to humans and consists of residues involved in the binding of metal ions | Mycobacterium tuberculosis |
| additional information | enzyme residues E57 and D251 are critical for catalysis, molecular modelling and mutational analysis. Determinants of abasic-site recognition, overview. Determinants of abasic-site recognition: the first three determinants, i.e. the base opposite the abasic site, the abasic ribose ring itself, and local distortions in the AP-site, do not play a role in MtbXthA, and in fact the enzyme exhibits robust endonucleolytic activity against single-stranded AP DNA also. Regarding the fourth determinant, conserved residues located near the active site, it is known that the catalytic-site of AP endonucleases is surrounded by conserved aromatic residues and intriguingly, the exact residues that are directly involved in abasic site recognition vary with the individual proteins. Y237, supported by Y137, mediates the formation of the MtbXthA-AP-DNA complex and AP-site incision. MtbXthA binds with high affinity to abasic sites in DNA e.g. to a 5'-FAM labelled duplex DNA substrate N1 that has an abasic site analogue, tetrahydrofuran (THF), incorporated into it. Homology modeling of MtbXthA using the structure of the Neisseria meningitidis protein (PDB ID 2JC4) as a template | Mycobacterium tuberculosis |
| physiological function | Mycobacterium tuberculosis AP-endonuclease/3'-5' exodeoxyribonuclease (MtbXthA) is an important player in DNA base excision repair. The enzyme has robust apurinic/apyrimidinic (AP) endonuclease activity, 3'-5' exonuclease, phosphatase, and phosphodiesterase activities. The enzyme functions as AP-endonuclease at high ionic environments, while the 3'-5' exonuclease activity is predominant at low ionic environments | Mycobacterium tuberculosis |
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