Rescuing DNA repair activity by rewiring the H-atom transfer pathway in the radical SAM enzyme, spore photoproduct lyase

Alhosna Benjdia, Korbinian Heil, Andreas Winkler, Thomas Carell, Ilme Schlichting
2014 Chemical Communications  
The radical SAM enzyme, spore photoproduct lyase, requires an H-atom transfer (HAT) pathway to catalyze DNA repair. By rational engineering, we demonstrate that it is possible to rewire its HAT pathway, a first step toward the development of novel catalysts based on the radical SAM enzyme scaffold. Spore photoproduct lyase (SP lyase) is a radical SAM enzyme catalyzing the repair of a unique thymidine dimer: 5-(a-thyminyl)-5,6-dihydrothymidine, a DNA damage encountered specifically in bacterial
more » ... cally in bacterial spores and commonly called the spore photoproduct (SP) (Scheme 1). 1, 2 In contrast to DNA photolyases (CPD and 6-4 photolyases), which use light energy and a flavin cofactor to catalyze the radicalbased repair of thymidine dimers, SP lyase requires an iron-sulfur cluster and S-adenosyl-L-methionine (SAM). 1,3 SP lyase generates the highly reactive 5 0 -deoxyadenosyl (5 0 -dA) radical which has been demonstrated to abstract the C6 proR-hydrogen atom of SP 4,5 inducing the formation of the 5-thyminyl-5,6-dihydrothymin-6-yl radical. After radical migration, the methylene bridge between the two nucleobase residues is cleaved (similar to what happens with DNA photolyases) 1 and a 3 0 -thymine allylic radical intermediate is likely formed. 6 Though, in contrast to DNA photolyases in which an electron from the repaired lesion is given back to the flavine cofactor to complete the catalytic cycle, SP lyase uses a complex and still unclear mechanism to regenerate the SAM cofactor. 1 We and others have established that SP lyase requires a critical cysteine residue to conclude the repair reaction. 5-8 The importance of this conserved residue (C141 in Bacillus subtilis) was established by mutagenesis studies showing that in vivo, C141 is critical for the viability of bacterial spores exposed to UV radiation, 8 while in vitro, substitution of C141 invariably led to the formation of DNA adducts (i.e. a sulfinic acid adduct). 6 We solved the crystal structure of SP lyase from Geobacillus thermodenitrificans (Gt) and discovered that this crucial cysteine residue (C140 in Gt) is located in close proximity to the SP lesion. 7 The position and the distance of this cysteine residue, relative to the a-methylene carbon atom of the 3 0 -thymine moiety (4.5 Å), led us to propose a function as ultimate H-atom donor to the DNA lesion and a key role in the ill-defined migration and control of radicals inside the enzyme active site. 7 C141 is strictly conserved among Bacilli species. However, despite the very high sequence homologies between SP lyases from Bacilli and Clostridia species (Fig. S1 , ESI †), clostridial SP lyases lack this functionally critical residue, forming thus a distinct sub-class of enzymes (Fig. S1 , ESI †). To investigate mechanistic and structural differences between these two subclasses of SP lyases, we built a structural model of the SP lyase from Clostridium acetobutilicum (Ca) which has been previously biochemically characterized. 9 The comparison of the active sites of the modeled clostridial SP lyase with the Gt enzyme showed that the architecture of the active sites, including the spatial orientation of residues involved in the binding of the DNA lesion, is conserved (Fig. S2, ESI †) . However, no cysteine residue in the clostridial SP lyase model superposed with the Scheme 1 Formation and repair of the spore photoproduct. † Electronic supplementary information (ESI) available: The atomic coordinates and structure factor amplitudes of the structures have been deposited in the Protein Data Bank with the accession numbers: 4RH0 and 4RH1. See ; Fax: +33 (0)1 34 65 24 62; Tel: +33 (0)1 34 65 24 33.
doi:10.1039/c4cc05158k pmid:25285338 fatcat:d7auti2lnvdg7jlkzjby5azwk4