Synthesis and Stereochemical Assignment of DNA Spore Photoproduct Analogues

Abstract

Investigation of the DNA repair process performed by the spore photoproduct (SP) lyase repair enzyme is strongly hampered by the lack of defined substrates needed for detailed enzymatic studies. The problem is particularly severe because the repair enzyme belongs to the class of strongly oxygen-sensitive radical (S)-adenosylmethionine (SAM) enzymes, which are notoriously difficult to handle. We report the synthesis of the spore photoproduct analogues 1 a and 1 b, which have open backbones and are diastereoisomers. In order to solve the problem of stereochemical assignment, two further derivatives 2 a and 2 b with closed backbones were prepared. The key step of the synthesis of 2 a/b is a metathesis-based macrocyclization that strongly increases the conformational rigidity of the synthetic spore photoproduct derivatives. NOESY experiments of the cyclic isomers furnished a clear cross-peak pattern that allowed the unequivocal assignment of the stereochemistry. The results were transferred to the data for isomers 1 a and 1 b, which were subsequently used for enzymatic-repair studies. These studies were performed with the novel spore photoproduct lyase repair enzyme from Geobacillus stearothermophilus. The studies showed an accordance with a recent investigation performed by us with the spore photoproduct lyase from Bacillus subtilis, in that only the S isomer 1 a is recognized and repaired. The ability to prepare a defined functioning substrate now paves the way for detailed enzymatic studies of the SP-lyase lesion recognition and repair process.

Structural insights into recognition and repair of UV-DNA damage by Spore Photoproduct Lyase, a radical SAM enzyme

“…For experimental phasing, two approaches were used: crystals of the native protein were soaked for 5 min in the cryoprotectant solution supplemented by 100 mM of 5-amino-2,4,6-triiodoisophthalic acid solution (Jena Bioscience, Jena, Germany) (16) and selenomethionine-substituted protein was crystallized and cryoprotected as described for the native protein. The substrate-bound SP lyase crystal structure was obtained by soaking crystals for 10 min in a solution containing 20 mM dinucleoside spore photoproduct (5 R –SP side synthesized as described previously (17) and shown in Supplementary Figure S1), the mother liquor, 3 mM dithiothreithol, 50 mM NaCl, 15 mM pyrophosphate, 14 mM hexametaphosphate (Sigma-Aldrich) and 15% (vol/vol) ethylene glycol.…”

“…The wild-type, reconstituted SP lyase (125 μM) was incubated with 5 mM dithiothreitol, 2 mM SAM, 250 μM SP side (5 R –SP side or 5 S –SP side synthesized as described previously (17) and shown in Supplementary Figure S1) in a Tris buffer (50 mM Tris–HCl pH 8 and 500 mM NaCl) and the repair reaction was started by adding 3 mM sodium dithionite. Control reactions were performed by omitting sodium dithionite.…”

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

“…Some earlier studies suggested that SPL recognizes and repairs the 5S-isomer (65,66), which has been corrected (67). In a recent SPL structure solved by Benjdia et al (68), the enzyme contains a dinucleoside 5R-SP, further confirming that the 5R-isomer is the SPL substrate.…”

Mechanistic studies of the radical SAM enzyme spore photoproduct lyase (SPL)

“…48,63,67 Besides using the traditional photochemical approach to generate the SP, synthetic chemistry have been developed to prepare the dinucleotide and dinucleoside SP as well as incorporate SP into the oligomer strand. 44,64,66–68,106 Although the syntheses are lengthy and the overall yields are low, SP preparation in milligram scale has become a realistic expectation. The progress in the SP synthesis resulted in the first structural characterizations of SP.…”