Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase

Abstract

Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a [3Fe-4S] + cluster accounting for 3-4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe-4S] + cluster is observed that accounts for 34-45% of total iron. Addition of S-adenosyl-methionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-Lmethionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 ± 0.6 nmol min −1 mg −1 , whereas no repair was observed for the 5S diastereomer.

NifEN-B complex of Azotobacter vinelandii is fully functional in nitrogenase FeMo cofactor assembly

“…The form of SAM used in the maturation assays is S-(5′-adenosyl)-L-methionine chloride. A SAM-induced decrease of the magnitude of the SAM-motifassociated S ¼ 1∕2 signal has been documented earlier (19) and could be explained by the oxidation of the unique SAM-motif ½Fe 4 S 4 cluster from the þ1 state to the EPR-silent, þ2 state upon the binding of SAM, which subsequently supplies electrons for the reductive cleavage of SAM (20). The simultaneous decrease in the signal intensity of the ½Fe 4 S 4 -type intermediate, therefore, reflects an immediate impact of the redox events at the SAMmotif on the electronic properties of the ½Fe 4 S 4 -intermediate -perhaps through an initial redox modification of the ½Fe 4 S 4 cluster, followed by a more complicated rearrangement of two ½Fe 4 S 4 modules into an 8Fe-cluster-both of which capable of rendering the cluster species in an EPR-silent state.…”

Spore Photoproduct Lyase: The Known, the Controversial, and the Unknown

“…However, other studies using smaller but chemically better-defined substrates have found much lower turnover numbers (72,(75)(76)(77)80). Most other in vitro SPL studies utilized dinucleotide or dinucleoside SP as substrate and found that the ratio between 5Ј-dA generated and SP repaired ranges between 1 and 2, suggesting that SAM is partially regenerated.…”

“…As the rate-limiting step for in vivo repair is likely to be damage recognition (90), the enzyme repair should be faster. However, the reported repair rate for dinucleotide SP TpT is only ϳ0.35 min Ϫ1 (24,72,80); the rate in duplex DNA may be even slower. Such slow rates cannot explain the fact that SPL plays a major role in SP repair in germinating spores.…”

“…Most other in vitro SPL studies utilized dinucleotide or dinucleoside SP as substrate and found that the ratio between 5Ј-dA generated and SP repaired ranges between 1 and 2, suggesting that SAM is partially regenerated. SPL activity using dinucleotide SP TpT as the substrate was reported to be ϳ0.3 min Ϫ1 by different groups (72,75,77,80), suggesting that the enzymes used were similarly active. Unpublished results from our laboratory, 3 however, found SPL to repair SP in a 20-nucleotide duplex prepared via DNA synthesis (81) at only 0.08 Ϯ 0.01 min Ϫ1 .…”

An Efficient Deprotection of N-Trimethylsilylethoxymethyl (SEM) Groups From Dinucleosides and Dinucleotides

“…[17–20] Our recent work on the synthesis and repair of spore photoproduct using spore photoproduct lyase provides direct evidence for the stereochemical requirements of spore photoproduct repair by spore product lyase (SPL), with only the 5 R -stereoisomers (dinucleosides/dinucleotide) acting as substrates (Figure 2). [21,22] Our need for pure SP substrate for mechanistic studies has led us to develop methods for the synthesis of stereochemically-defined dinucleoside and dinucleotide SP substrates. [21–23] The complete multistep synthesis of 5 R and 5 S -spore photoproducts from thymidine involves the condensation of dihydrothymidine and thymidine, selective deprotection of triethylsilyl groups, [23] phosphorylation at the 3′ hydroxyl of dihydrothymidine and phosphodiester bond formation between dihydrothymidine and thymidine.…”