Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase.
DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.
Hamm-Alvarez
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Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Sancar
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A
Rajagopalan
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KV
D001894:Borohydrides; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002384:Catalysis; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000032:analysis / Q000378:metabolism; D008027:Light; D008190:Lyases / Q000502:physiology*; D010782:Photolysis; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates / Q000378:metabolism / Q000502:physiology*
D001894:Borohydrides; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates; C037860:5,10-methylenetetrahydrofolate polyglutamate; C025364:sodium borohydride; D005492:Folic Acid; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase