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Literature summary for 4.1.99.14 extracted from

  • Li, L.
    Mechanistic studies of the radical SAM enzyme spore photoproduct lyase (SPL) (2012), Biochim. Biophys. Acta, 1824, 1264-1277.
    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 N- or C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) Bacillus subtilis

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ utilizes a tricysteine CXXXCXXC motif to harbor a [4Fe-4S] cluster 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 N- or C-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity 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 catalyzes the spore photoproduct repair reaction via a radical mediated direct reverse mechanism. At the 1+ oxidation state, the [4Fe-4S] cluster provides an electron to the S-adenosyl-L-methionine, which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the C-S bond associated with the sulfonium ion in S-adenosyl-L-methionine, generating a reactive 5'-deoxyadenosyl radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of spore photoproduct to initiate the repair process. The resulting spore photoproduct radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired thymidylyl-(3'->5')-thymidylate. Cys141 is involved in the catalytic mechanism as the potential H atom donor to the thymine methyl radical, reaction mechanism, detailed overview. A a thiyl radical is subsequently generated on Cys141 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 i.e. spore photoproduct, an in situ monomerization Bacillus subtilis thymidylyl-(3'->5')-thymidylate
-
 ?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
-
 Bacillus subtilis 168 thymidylyl-(3'->5')-thymidylate
-
 ?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine i.e. spore photoproduct, an in situ monomerization Bacillus subtilis 168 thymidylyl-(3'->5')-thymidylate
-
 ?
additional information the enzyme recognizes and repairs a range of spore photoproduct containing DNA substrates, ranging from dinucleotide and dinucleoside spore photoproduct to spore photoproduct containing single- and double-stranded DNA. The fastest reaction rate employs a single-stranded spore photoproduct containing GGSPGG 6-mer as the substrate, while the double-stranded spore photoproduct-containing plasmid DNA also supports a fast repair reaction Bacillus subtilis ?
-
 ?
additional information the enzyme recognizes and repairs a range of spore photoproduct containing DNA substrates, ranging from dinucleotide and dinucleoside spore photoproduct to spore photoproduct containing single- and double-stranded DNA. The fastest reaction rate employs a single-stranded spore photoproduct containing GGSPGG 6-mer as the substrate, while the double-stranded spore photoproduct-containing plasmid DNA also supports a fast repair reaction Bacillus subtilis 168 ?
-
 ?

Synonyms

Synonyms Comment Organism
SPL
-
 Bacillus subtilis

Cofactor

Cofactor Comment Organism Structure
S-adenosyl-L-methionine a radical S-adenosyl-L-methionine enzyme, S-adenosyl-L-methionine is suggested to be regenerated at the end of each catalytic cycle, and only a catalytic amount of S-adenosyl-L-methionine is needed in the enzyme reaction. The H atom source for the back donation step is suggested to be a cysteine residue 141, and the H-atom transfer reaction leaves a thiyl radical behind on the protein. This thiyl radical thus must participate in the S-adenosyl-L-methionine regeneration process, the thiyl radical abstracts an H atom from the 5'-deoxyadenosyl radical to regenerate S-adenosyl-L-methionine Bacillus subtilis

General Information

General Information Comment Organism
evolution the enzyme is a member of the so-called radical SAM superfamily, which is defined by the characteristic CXXXCXXC motif. The three cysteine residues serve as ligands respectively for three irons in the [4Fe-4S] cluster, with the fourth iron being coordinated by the S-adenosylmethionine in a bi-dentate manner, with its amino and carboxylate moieties serving as the fourth and fifth ligands to the cluster. SPL and the DNA photolyase, EC 4.1.99.3, show amino acid sequence homolgy and might have descended from a common ancestral protein Bacillus subtilis
additional information the enzyme protects at least 9 nucleotides in the spore photoproduct containing DNA strand with 5 nucleotides 3' to and 2 nucleotides 5' to the spore photoproduct damage, suggesting that the phosphates included in this region may be involved in the binding interaction with the enzyme Bacillus subtilis
physiological function spore photoproduct lyase repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct at the bacterial early germination phase. Spore photoproduct is the exclusive DNA photo-damage product in bacterial endospores. Its generation and swift repair by the enzyme are responsible for the spores' extremely high UV resistance Bacillus subtilis