Cloned (Comment) | Organism |
---|---|
DNA constructs encoding mutant Endo IV enzymes are generated with the megaprimer PCR technique47 using a wild-type nfo gene sequence as template. The PCR products are purified, digested and subcloned in pET24. Hexahistidine-tagged wild-type Endo IV and mutants are subcloned in vector pET28a. The double mutants (Y72A/E261Q and R37A/E261Q) are obtained by Megaprimer mutagenesis on the E261Q mutant followed by subcloning of the PCR product in frame with an N-terminal His6-sequence in pET28a. Wild-type and mutant enzymes are produced and purified using an nfo-negative strain BW565DE3 transformed with pET24-Endo IV constructs | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
Crystals are grown by vapor diffusion. The 1.10-A resolution DNA-free and the 2.45-A resolution DNA-substrate complex structures capture substrate stabilization by Arg37 and reveal a distorted Zn3-ligand arrangement that reverts, after catalysis, to an ideal geometry suitable to hold rather than release cleaved DNA product. Coordinates and structure factors for the three structures (E261Q-phosphate, E261Q-AP DNA and Y72A-AP DNA) have been deposited with accession codes 2NQH, 2NQJ and 2NQ9 | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
D179N | mild (10fold reduction) activity | Escherichia coli |
D229N | reduction in activity | Escherichia coli |
E145Q | complete loss of catalytic activity | Escherichia coli |
E261Q | catalytically inactive mutant, Glu261 is essential to catalysis | Escherichia coli |
H109N | mild (10fold reduction) activity | Escherichia coli |
H182N | reduction in activity | Escherichia coli |
H216N | reduction in activity | Escherichia coli |
H231N | reduction in activity | Escherichia coli |
H69N | reduction in activity | Escherichia coli |
R37A | strong positive effect on catalytic activity | Escherichia coli |
R37A/E261Q | shows a three-fold decrease in AP-DNA binding affinity (Kd 340 nM) | Escherichia coli |
Y72A | Tyr72 is important for the stability of the enzyme-substrate complex | Escherichia coli |
Y72A/E261Q | double mutant shows a dissociation constant of Kd 97 nM | Escherichia coli |
Y72F | Tyr72 is important for the stability of the enzyme-substrate complex | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
AP-DNA | not detected in mutant E261Q in rAP-containing oligonucleotide cleavage assay | Escherichia coli | |
0.000088 | - |
AP-DNA | wild-type in rAP-containing oligonucleotide cleavage assay | Escherichia coli | |
0.00012 | - |
AP-DNA | mutant Y72F in rAP-containing oligonucleotide cleavage assay | Escherichia coli | |
0.0075 | - |
AP-DNA | mutant Y72A in rAP-containing oligonucleotide cleavage assay | Escherichia coli | |
0.011 | - |
AP-DNA | mutant R37A in rAP-containing oligonucleotide cleavage assay | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mn2+ | divalent metal content | Escherichia coli | |
additional information | equal or higher activity for Zn2+ or Mn2+- containing Endo IV suggests that one site may favor Mn2+ over Zn2+ | Escherichia coli | |
Zn2+ | Divalent metal content, Zn3-site mutations result in major activity loss, whereas Zn1 and Zn2 ligand mutations cause low to severe loss of catalytic efficiency. In the DNA-free wild-type enzyme structure, two metal ions (Zn1 and Zn2) are partially buried from solvent and bind a bridging hydroxide anion. The third metal ion (Zn3) is mostly solvent accessible, is distant from Zn1 and Zn2 and ligates a tightly bound water molecule to complete its coordination shell | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
AP-DNA | Escherichia coli | - |
fragments of DNA | - |
? | |
AP-DNA | Escherichia coli BW565DE3 | - |
fragments of DNA | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Escherichia coli BW565DE3 | - |
- |
- |
Purification (Comment) | Organism |
---|---|
by nickel-nitrilotriacetic acid affinity chromatography | Escherichia coli |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
Activity is determined with phage T4 AP DNA endonuclease assay agrees with divalent metal (Mn2+ and Zn2+) content measured for purified His-tagged Endo IV wild-type and mutant proteins, except for E261Q and to a lesser extent E145Q, which are adjacent in the active site. Mutations of the first-shell metal-ion binding residues resulted in mild (10fold reduction activity, H109N and D179N), severe (100-2600fold reduction in activity, H69N, H182N, D229N, H216N and H231N) or complete loss (E145Q, E261Q) of catalytic activity in both the T4 AP-DNA endonuclease assay and the AP-oligonucleotide cleavage assay | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
AP-DNA | - |
Escherichia coli | fragments of DNA | - |
? | |
AP-DNA | - |
Escherichia coli BW565DE3 | fragments of DNA | - |
? |
Synonyms | Comment | Organism |
---|---|---|
APE1 | - |
Escherichia coli |
endonuclease IV | an abasic or apurinic-apyrimidinic endonuclease superfamily crucial for DNA base excision repair | Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
AP-DNA | not detected in mutant E261Q | Escherichia coli | |
72 | - |
AP-DNA | mutant Y72A | Escherichia coli | |
90 | - |
AP-DNA | wild-type | Escherichia coli | |
354 | - |
AP-DNA | mutant Y72F | Escherichia coli | |
7680 | - |
AP-DNA | mutant R37A | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.1 | - |
assay at | Escherichia coli |