Literature summary for 4.2.99.18 extracted from

Garcin, E.D.; Hosfield, D.J.; Desai, S.A.; Haas, B.J.; Bjoeras, M.; Cunningham, R.P.; Tainer, J.A.
DNA apurinic-apyrimidinic site binding and excision by endonuclease IV (2008), Nat. Struct. Mol. Biol., 15, 515-522.

Search for more literature for 4.2.99.18

Cloned(Commentary)

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

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 (Commentary)

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

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

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 [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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

Metals/Ions	Comment	Organism
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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Escherichia coli	-	-	-
Escherichia coli BW565DE3	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
by nickel-nitrilotriacetic acid affinity chromatography	Escherichia coli

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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

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 [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
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

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8.1	-	assay at	Escherichia coli