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

Abstract

Spore photoproduct lyase (SPL) repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct or SP at the bacterial early germination phase. SP is the exclusive DNA photo-damage product in bacterial endospores; its generation and swift repair by SPL are responsible for the spores’ extremely high UV resistance. The early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair the SP in the absence of light. The research in the past decade further established SPL as a radical SAM enzyme, which utilizes a tri-cysteine CXXXCXXC motif to harbor a [4Fe-4S] cluster. At the 1+ oxidation state, the cluster provides an electron to the S-adenosylmethionine (SAM), 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 SAM, generating a reactive 5′-deoxyadenosyl (5′-dA) radical. This 5′-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. SAM is suggested to be regenerated at the end of each catalytic cycle; and only a catalytic amount of SAM is needed in the SPL reaction. The H atom source for the back donation step is suggested to be a cysteine residue (C141 in B. subtilis SPL), and the H-atom transfer reaction leaves a thiyl radical behind on the protein. This thiyl radical thus must participate in the SAM regeneration process; however how the thiyl radical abstracts an H atom from the 5′-dA to regenerate SAM is unknown. This paper reviews and discusses the history and the latest progress in the mechanistic elucidation of SPL. Despite some recent breakthroughs, more questions are raised in the mechanistic understanding of this intriguing DNA repair enzyme.

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA

“…In phages such as MP1412, YuA, M6 the equivalent radical-SAM domain occurs independently of the thymidylate synthase-like domain and is catalytically active. It could function downstream of the clade-2 phosphorylase to generate thymine from the methylene intermediate using a reaction similar that used by SplB to restore thymine from photo-dimers (91). It is also accompanied by a further gene coding for a pyridoxal 5′-Phosphate Dependent (PLPDE) superfamily enzyme that could potentially function as an aminotransferase needed to synthesize the amino-group-containing moiety that is ligated to the methyl group of thymine (Figure 4) (92).…”

Unusually Large Deuterium Discrimination during Spore Photoproduct Formation

“…4 As much as 8% of the total genomic thymine residues can be converted to SPs, 5 which is repaired by spore photoproduct lyase (SPL) during spore germination, ensuring their normal life cycle is not disrupted. 4b,4d,4e,6 These SPs, if unrepaired, prove lethal to the germinated spores.…”

The Enzyme-Mediated Direct Reversal of a Dithymine Photoproduct in Germinating Endospores

“…proved that the radioactivity disappearing from SP appeared to be stoichiometrically recovered in thymine [21], further supporting this direct-reversal hypothesis. The property of SP and the strong UV resistance it brings to bacterial spores have been covered by several excellent reviews in the past several years [1,9,10,29–32]. In this paper, we will focus on the latest progress in the mechanistic elucidation of SPL.…”