Literature summary for 4.1.99.14 extracted from

Benjdia, A.; Heil, K.; Barends, T.R.; Carell, T.; Schlichting, I.
Structural insights into recognition and repair of UV-DNA damage by spore photoproduct lyase, a radical SAM enzyme (2012), Nucleic Acids Res., 40, 9308-9318.

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Cloned(Commentary)

Cloned (Comment)	Organism
gene GTNG_2348, expression of His6-tagged native and selenomethionine-labeled wild-type enzyme and His6-tagged mutant enzymes in Escherichia coli strain BL21(DE3)	Geobacillus thermodenitrificans

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified wild-type and mutant enzymes in complex with the S-adenosyl-L-methionine cofactor and the [4Fe-4S] cluster, hanging drop vapor diffusion method, using 70 mM octanoyl-N-hydroxyethylglucamide and a reservoir solution containing 200 mM lithium sulfate, 100 mM Tris-HCl, pH 9.0, and 19-27% w/v PEG 8000., 20°C, cryoprotection of crystals by 3 mM dithiothreithol, 500 mM NaCl, and 15% v/v ethylene glycol in mother liquor, X-ray diffraction structure determination and analysis at 2.0-2.1 A resolution	Geobacillus thermodenitrificans

Crystallization (Comment)

Organism

purified wild-type and mutant enzymes in complex with the S-adenosyl-L-methionine cofactor and the [4Fe-4S] cluster, hanging drop vapor diffusion method, using 70 mM octanoyl-N-hydroxyethylglucamide and a reservoir solution containing 200 mM lithium sulfate, 100 mM Tris-HCl, pH 9.0, and 19-27% w/v PEG 8000., 20°C, cryoprotection of crystals by 3 mM dithiothreithol, 500 mM NaCl, and 15% v/v ethylene glycol in mother liquor, X-ray diffraction structure determination and analysis at 2.0-2.1 A resolution

Geobacillus thermodenitrificans

Protein Variants

Protein Variants	Comment	Organism
C140A	site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but 2.5fold reduced repair activity	Geobacillus thermodenitrificans
C140S	site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but reduced repair activity	Geobacillus thermodenitrificans

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	the enzyme uses a [4Fe-4S]1+ cluster and S-adenosyl-L-methionine to initiate the repair reaction	Geobacillus thermodenitrificans

Organism

Organism	UniProt	Comment	Textmining
Geobacillus thermodenitrificans	A4IQU1	gene GTNG_2348	-
Geobacillus thermodenitrificans NG80-2	A4IQU1	gene GTNG_2348	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type, selenomethionine-labeled, and mutant His6-tagged enzymes from Escherichia coli strain BL21(DE3) by nickel and heparin affinity chromatography	Geobacillus thermodenitrificans

Reaction

Reaction	Comment	Organism	Reaction ID
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)	the enzyme catalyzes the spore photoproduct repair reaction and combines specific features of radical S-adenosyl-L-methionine and DNA repair enzymes to enable a complex radical-based repair reaction to take place	Geobacillus thermodenitrificans	a

Synonyms

Synonyms	Comment	Organism
SP lyase	-	Geobacillus thermodenitrificans

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
22	-	assay at room temperature	Geobacillus thermodenitrificans

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Geobacillus thermodenitrificans

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	a radical S-adenosyl-L-methionine enzyme that uses a [4Fe-4S]1+ cluster and S-adenosyl-L-methionine to initiate the repair reaction	Geobacillus thermodenitrificans

General Information

General Information	Comment	Organism
evolution	unlike DNA photolyases, EC 4.1.99.3, SP lyase belongs to the emerging superfamily of radical S-adenosyl-L-methionine (SAM) enzymes	Geobacillus thermodenitrificans
additional information	active site structure, DNA lesion recognition, and substrate binding which involve a beta-hairpin structure, overview. S-adenosyl-L-methionine and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the alpha-thyminyl radical moiety, respectively. Structure comparison of wild-type and C140 mutant enzymes, overview	Geobacillus thermodenitrificans