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Literature summary for 4.1.99.14 extracted from

  • Benjdia, A.; Heil, K.; Winkler, A.; Carell, T.; Schlichting, I.
    Rescuing DNA repair activity by rewiring the H-atom transfer pathway in the radical SAM enzyme, spore photoproduct lyase (2014), Chem. Commun. (Camb.), 50, 14201-14204 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene GTNG_2348, sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Geobacillus thermodenitrificans

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant reconstituted C140A/S76C mutant SP lyase, hanging drop vapor diffusion method, the enzyme is incubated with Se-SAM, crystallization in the presence of 70 mM octanoyl-N-hydroxyethylglucamide and mixing with a reservoir solution containing 200 mM lithium sulfate, 100 mM Tris-HCl, pH 9.0, and 19-27% w/v PEG 8000 under anaerobic conditions, 20°C, X-ray diffraction structure determination and analysis at 2.1-2.6 A resolution Geobacillus thermodenitrificans

Protein Variants

Protein Variants Comment Organism
C140A site-directed mutagenesis Geobacillus thermodenitrificans
C140A/S76C site-directed mutagenesis Geobacillus thermodenitrificans
additional information by rational engineering, the enzyme's HAT pathway is rewired. Possible development of improved catalysts based on the radical SAM enzyme scaffold Geobacillus thermodenitrificans

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ [4Fe-4S] cluster Geobacillus thermodenitrificans

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine Geobacillus thermodenitrificans in double-helical DNA thymidylyl-(3'->5')-thymidylate
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine Geobacillus thermodenitrificans NG80-2 in double-helical DNA thymidylyl-(3'->5')-thymidylate
-
?

Organism

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

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and heparin affinity chromatography. The iron-sulfur cluster of the purified proteins is then reconstituted Geobacillus thermodenitrificans

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine in double-helical DNA Geobacillus thermodenitrificans thymidylyl-(3'->5')-thymidylate
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine in double-helical DNA Geobacillus thermodenitrificans NG80-2 thymidylyl-(3'->5')-thymidylate
-
?
additional information activity of the reconstituted wild-type enzyme and the enzyme mutants is measured using a 13-mer oligonucleotide containing the dinucleoside SP (5'-CAGCGGT-TGCAGG-3') as substrate, mass spectrometric poduct analysis, overview. The repair of the SP containing DNA leads to two oligonucleotides: the 7 mer (5'-CAGCGGT-3') and the 6 mer (5'-TGCAGG-3') Geobacillus thermodenitrificans ?
-
?
additional information activity of the reconstituted wild-type enzyme and the enzyme mutants is measured using a 13-mer oligonucleotide containing the dinucleoside SP (5'-CAGCGGT-TGCAGG-3') as substrate, mass spectrometric poduct analysis, overview. The repair of the SP containing DNA leads to two oligonucleotides: the 7 mer (5'-CAGCGGT-3') and the 6 mer (5'-TGCAGG-3') Geobacillus thermodenitrificans NG80-2 ?
-
?

Subunits

Subunits Comment Organism
More enzyme secondary structure comparisons using the crystal structures of the substrate-free wild-type SP lyase from Geobacillus thermodenitrificans, PDB ID 4FHC, of enzyme mutant C140A mutant (PDB code 4FHF), and the enzyme double mutant SP lyase Geobacillus thermodenitrificans

Synonyms

Synonyms Comment Organism
Gt SP lyase
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Geobacillus thermodenitrificans
GTNG_2348
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Geobacillus thermodenitrificans
SP lyase
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Geobacillus thermodenitrificans

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at 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 SPL is a radical SAM enzyme Geobacillus thermodenitrificans
[4Fe-4S] cluster
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Geobacillus thermodenitrificans

General Information

General Information Comment Organism
physiological function the radical SAM enzyme, spore photoproduct lyase, requires an H-atom transfer (HAT) pathway to catalyze DNA repair Geobacillus thermodenitrificans