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Literature summary for 4.1.99.14 extracted from

  • Yang, L.; Nelson, R.S.; Benjdia, A.; Lin, G.; Telser, J.; Stoll, S.; Schlichting, I.; Li, L.
    A radical transfer pathway in spore photoproduct lyase (2013), Biochemistry, 52, 3041-3050.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 4.1.99.14

Cloned(Commentary)

Cloned (Comment) Organism
gene splB, overexpression of His6-tagged wild-type and mutant enzymes and co-expression with plasmid pDB1282, in Escherichia coli strain BL21(DE3) Bacillus subtilis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant His6-tagged mutant enzyme Y98F, with reconstituted iron-sulfur cluster, hanging drop vapor diffusion method, using 70 mM octanoyl-N-hydroxyethylglucamide, 200 mM lithium sulfate, 100 mM Tris-HCl, pH 9.0 and 19-27% w/v PEG 8000, X-ray diffraction structure determination and analysis Bacillus subtilis

Protein Variants

Protein Variants Comment Organism
C141A site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme Bacillus subtilis
additional information compared to tyrosine, phenylalanine retains the aromatic ring, but does not support the radical propagation reaction due to the loss of the OH moiety. [4Fe-4S] clusters remains intact in the Y->F mutants Bacillus subtilis
Y97A site-directed mutagenesis, the mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme Bacillus subtilis
Y97A/Y99A site-directed mutagenesis, inactive mutant, the Y97 mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage Bacillus subtilis
Y97F site-directed mutagenesis, the mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme Bacillus subtilis
Y98A site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme Bacillus subtilis
Y98F site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme Bacillus subtilis
Y99A site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme Bacillus subtilis
Y99F site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme Bacillus subtilis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state kinetics and competitive kinetic isotope effects of recombinant wild-type and mutant enzymes, overview Bacillus subtilis

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ the enzyme uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine generating a catalytic 5'-deoxyadenosyl radical Bacillus subtilis

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 Bacillus subtilis
-
 thymidylyl-(3'->5')-thymidylate
-
 ?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine Bacillus subtilis 168
-
 thymidylyl-(3'->5')-thymidylate
-
 ?

Organism

Organism UniProt Comment Textmining
Bacillus subtilis
-
 gene splB
-
Bacillus subtilis 168
-
 gene splB
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and cation exchange chromatography Bacillus subtilis

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 is a radical S-adenosyl-L-methionine enzyme, which uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine, generating a catalytic 5'-deoxyadenosyl radical. This in turn abstracts an H atom from spore product, generating an spore product radical that undergoes beta scission to form a repaired 5'-thymine and a 3'-thymine allylic radical. A conserved cysteine donates an H atom to the thymine radical, resulting in a putative thiyl radical. Two conserved tyrosines are also critical in enzyme catalysis. One, Y99, is downstream of the cysteine, suggesting that the enzyme uses a hydrogen atom transfer pathway with a pair of cysteine-tyrosine residues to regenerate S-adenosyl-L-methionine. The other tyrosine, Y97, has a structural role to facilitate S-adenosyl-L-methionine binding. It may also contribute to the S-adenosyl-L-methionine regeneration process by interacting with the putative Y99 radical and/or 5-dA radical intermediates to lower the energy barrier for the second H-abstraction step. Irreversible first step and tightly coupled radical relay mechanism Bacillus subtilis
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
-
 Bacillus subtilis thymidylyl-(3'->5')-thymidylate
-
 ?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
-
 Bacillus subtilis 168 thymidylyl-(3'->5')-thymidylate
-
 ?

Synonyms

Synonyms Comment Organism
SPL
-
 Bacillus subtilis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
22
-
 assay at room temperature Bacillus subtilis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Bacillus subtilis

Cofactor

Cofactor Comment Organism Structure
S-adenosyl-L-methionine a radical S-adenosyl-L-methionine enzyme that uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine generating a catalytic 5'-deoxyadenosyl radical Bacillus subtilis

General Information

General Information Comment Organism
evolution the enzyme is the first member of the radical SAM superfamily (comprising more than 44000 members) to bear a catalytically operating hydrogen atom transfer chain that is essential for S-adenosyl-L-methionine regeneration after the catalytic cycle Bacillus subtilis
physiological function spore photoproduct lyase repairs a covalent UV-induced thymine dimer, spore photoproduct, in germinating endospores and is responsible for strong UV resistance of endospores Bacillus subtilis