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Literature summary extracted from

  • Khanam, T.; Shukla, A.; Rai, N.; Ramachandran, R.
    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 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.1.11.2 recombinant expression of wild-type and mutant enzymes Mycobacterium tuberculosis
4.2.99.18 recombinant expression of wild-type and mutant enzymes Mycobacterium tuberculosis

Protein Variants

EC Number Protein Variants Comment Organism
3.1.11.2 D251A site-directed mutagenesis, the mutant shows a 3fold decline in the endonucleolytic cleavage activity Mycobacterium tuberculosis
3.1.11.2 E57A site-directed mutagenesis, the mutant shows a 2fold decline in the endonucleolytic cleavage activity Mycobacterium tuberculosis
3.1.11.2 E57A/D251A site-directed mutagenesis, the double mutant shows no 3'-5' exonuclease activity Mycobacterium tuberculosis
3.1.11.2 F242S site-directed mutagenesis, the mutant shows slightly increased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
3.1.11.2 W235S site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
3.1.11.2 Y137S site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
3.1.11.2 Y234S site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
3.1.11.2 Y234S/W235S site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
3.1.11.2 Y237S site-directed mutagenesis, the mutant shows no 3'-5' exonuclease activity Mycobacterium tuberculosis
4.2.99.18 D251A site-directed mutagenesis, the mutant shows a 3fold decline in the endonucleolytic cleavage activity Mycobacterium tuberculosis
4.2.99.18 E57A site-directed mutagenesis, the mutant shows a 2fold decline in the endonucleolytic cleavage activity Mycobacterium tuberculosis
4.2.99.18 E57A/D251A site-directed mutagenesis, the double mutant shows no 3'-5' exonuclease activity Mycobacterium tuberculosis
4.2.99.18 F242S site-directed mutagenesis, the mutant shows increased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
4.2.99.18 W235S site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
4.2.99.18 Y137S site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
4.2.99.18 Y234S site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
4.2.99.18 Y234S/W235S site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type Mycobacterium tuberculosis
4.2.99.18 Y237S site-directed mutagenesis, the mutant shows no 3'-5' exonuclease activity Mycobacterium tuberculosis

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.1.11.2 NaCl addition of more than 200 mM salt, strongly inhibits the endonuclease activity Mycobacterium tuberculosis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.1.11.2 additional information
-
 additional information steady-state Michaelis-Menten kinetic analysis Mycobacterium tuberculosis
3.1.11.2 0.000014
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant F242S Mycobacterium tuberculosis
3.1.11.2 0.000015
-
 N1 duplex DNA pH 7.8, 37°C, recombinant wild-type enzyme Mycobacterium tuberculosis
3.1.11.2 0.000015
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant D251A Mycobacterium tuberculosis
3.1.11.2 0.000016
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant E57A Mycobacterium tuberculosis
3.1.11.2 0.000016
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant Y234S/W235S Mycobacterium tuberculosis
3.1.11.2 0.000018
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant W235S Mycobacterium tuberculosis
3.1.11.2 0.000022
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant Y234S Mycobacterium tuberculosis
3.1.11.2 0.000064
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant Y137S Mycobacterium tuberculosis
4.2.99.18 additional information
-
 additional information steady-state Michaelis-Menten kinetic analysis Mycobacterium tuberculosis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.1.11.2 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
3.1.11.2 NaCl MtbXthA exhibits an increase in AP site incision activity in a salt-dependent manner Mycobacterium tuberculosis
4.2.99.18 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
4.2.99.18 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
4.2.99.18 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

EC Number Organism UniProt Comment Textmining
3.1.11.2 Mycobacterium tuberculosis P96273
-
-
3.1.11.2 Mycobacterium tuberculosis ATCC 25618 P96273
-
-
3.1.11.2 Mycobacterium tuberculosis H37Rv P96273
-
-
4.2.99.18 Mycobacterium tuberculosis P96273
-
-
4.2.99.18 Mycobacterium tuberculosis ATCC 25618 P96273
-
-
4.2.99.18 Mycobacterium tuberculosis H37Rv P96273
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.1.11.2 recombinant wild-type and mutant enzymes Mycobacterium tuberculosis
4.2.99.18 recombinant wild-type and mutant enzymes Mycobacterium tuberculosis

Source Tissue

EC Number Source Tissue Comment Organism Textmining
4.2.99.18 additional information XthA is expressed constitutively Mycobacterium tuberculosis
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.1.11.2 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. Analysis of the 3? phosphatase and 3' phosphodiesterase activities of MtbXthA with a DNA substrate. Enzyme substrate specificity analysis of 3'-5' exonuclease activity of MtbXthA using fluorescently labelled ssDNA X3, 3'-overhanging heteroduplex DNA X4, 3'-blunt DNA duplex X5, 3'-recessed heteroduplexDNA X1, and nicked DNA X2, detailed overview. MtbXthA exhibits the strongest exonuclease activity on a 3'-recessed end containing duplex DNA. No significant differences in the excision of the 16 3'-recessed duplex DNA substrates are found under the reaction conditions. These results show that MtbXthA does not differentiate between matched and mismatched base pairs. MtbXthA can effectively remove 3'-blocking termini Mycobacterium tuberculosis ?
-
 ?
3.1.11.2 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. Analysis of the 3? phosphatase and 3' phosphodiesterase activities of MtbXthA with a DNA substrate. Enzyme substrate specificity analysis of 3'-5' exonuclease activity of MtbXthA using fluorescently labelled ssDNA X3, 3'-overhanging heteroduplex DNA X4, 3'-blunt DNA duplex X5, 3'-recessed heteroduplexDNA X1, and nicked DNA X2, detailed overview. MtbXthA exhibits the strongest exonuclease activity on a 3'-recessed end containing duplex DNA. No significant differences in the excision of the 16 3'-recessed duplex DNA substrates are found under the reaction conditions. These results show that MtbXthA does not differentiate between matched and mismatched base pairs. MtbXthA can effectively remove 3'-blocking termini Mycobacterium tuberculosis H37Rv ?
-
 ?
3.1.11.2 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. Analysis of the 3? phosphatase and 3' phosphodiesterase activities of MtbXthA with a DNA substrate. Enzyme substrate specificity analysis of 3'-5' exonuclease activity of MtbXthA using fluorescently labelled ssDNA X3, 3'-overhanging heteroduplex DNA X4, 3'-blunt DNA duplex X5, 3'-recessed heteroduplexDNA X1, and nicked DNA X2, detailed overview. MtbXthA exhibits the strongest exonuclease activity on a 3'-recessed end containing duplex DNA. No significant differences in the excision of the 16 3'-recessed duplex DNA substrates are found under the reaction conditions. These results show that MtbXthA does not differentiate between matched and mismatched base pairs. MtbXthA can effectively remove 3'-blocking termini Mycobacterium tuberculosis ATCC 25618 ?
-
 ?
3.1.11.2 N1 duplex DNA + H2O 5'-FAM peptide-labelled AP DNA duplex substrate Mycobacterium tuberculosis ?
-
 ?
3.1.11.2 N1 duplex DNA + H2O 5'-FAM peptide-labelled AP DNA duplex substrate Mycobacterium tuberculosis H37Rv ?
-
 ?
3.1.11.2 N1 duplex DNA + H2O 5'-FAM peptide-labelled AP DNA duplex substrate Mycobacterium tuberculosis ATCC 25618 ?
-
 ?
4.2.99.18 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 ?
-
 ?
4.2.99.18 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 ?
-
 ?
4.2.99.18 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 ?
-
 ?
4.2.99.18 N1 duplex DNA substrate + H2O 5'-FAM peptide-labelled AP DNA duplex substrate Mycobacterium tuberculosis ?
-
 ?
4.2.99.18 N1 duplex DNA substrate + H2O 5'-FAM peptide-labelled AP DNA duplex substrate Mycobacterium tuberculosis H37Rv ?
-
 ?
4.2.99.18 N1 duplex DNA substrate + H2O 5'-FAM peptide-labelled AP DNA duplex substrate Mycobacterium tuberculosis ATCC 25618 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.1.11.2 3'-5' exonuclease III
-
 Mycobacterium tuberculosis
3.1.11.2 AP-endonuclease/3'-5'exodeoxyribonuclease
-
 Mycobacterium tuberculosis
3.1.11.2 More cf. EC 4.2.99.18, AP-endonuclease Mycobacterium tuberculosis
3.1.11.2 MtbXthA
-
 Mycobacterium tuberculosis
3.1.11.2 xthA
-
 Mycobacterium tuberculosis
4.2.99.18 AP endonuclease VI UniProt Mycobacterium tuberculosis
4.2.99.18 AP-endonuclease
-
 Mycobacterium tuberculosis
4.2.99.18 AP-endonuclease/3'-5'exodeoxyribonuclease
-
 Mycobacterium tuberculosis
4.2.99.18 More cf. EC 3.1.11.2, 3'-5' exonuclease III Mycobacterium tuberculosis
4.2.99.18 MtbXthA
-
 Mycobacterium tuberculosis
4.2.99.18 xthA
-
 Mycobacterium tuberculosis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.1.11.2 37
-
 assay at Mycobacterium tuberculosis
4.2.99.18 37
-
 assay at Mycobacterium tuberculosis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.1.11.2 0.001
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant E57A Mycobacterium tuberculosis
3.1.11.2 0.002
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant D251A Mycobacterium tuberculosis
3.1.11.2 0.008
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant Y137S Mycobacterium tuberculosis
3.1.11.2 0.01
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant Y234S/W235S Mycobacterium tuberculosis
3.1.11.2 0.012
-
 N1 duplex DNA pH 7.8, 37°C, recombinant wild-type enzyme Mycobacterium tuberculosis
3.1.11.2 0.012
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant F242S Mycobacterium tuberculosis
3.1.11.2 0.012
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant W235S Mycobacterium tuberculosis
3.1.11.2 0.012
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant Y234S Mycobacterium tuberculosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.1.11.2 7.8
-
 assay at Mycobacterium tuberculosis
4.2.99.18 7.8
-
 assay at Mycobacterium tuberculosis

General Information

EC Number General Information Comment Organism
3.1.11.2 additional information enzyme residues E57 and D251 are critical for catalysis, molecular modelling and mutational analysis. Determinants of abasic-site recognition, overview. Homology modeling of MtbXthA using the structure of the Neisseria meningitidis protein (PDB ID 2JC4) as a template Mycobacterium tuberculosis
3.1.11.2 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 an AP-endonuclease at high ionic environments, while the 3'-5' exonuclease activity is predominant at low ionic environments Mycobacterium tuberculosis
4.2.99.18 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
4.2.99.18 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
4.2.99.18 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

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.1.11.2 60
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant E57A Mycobacterium tuberculosis
3.1.11.2 110
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant Y137S Mycobacterium tuberculosis
3.1.11.2 130
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant D251A Mycobacterium tuberculosis
3.1.11.2 550
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant Y234S Mycobacterium tuberculosis
3.1.11.2 630
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant Y234S/W235S Mycobacterium tuberculosis
3.1.11.2 650
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant W235S Mycobacterium tuberculosis
3.1.11.2 800
-
 N1 duplex DNA pH 7.8, 37°C, recombinant wild-type enzyme Mycobacterium tuberculosis
3.1.11.2 880
-
 N1 duplex DNA pH 7.8, 37°C, recombinant mutant F242S Mycobacterium tuberculosis