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

ABSTRACT: Bacterial spores possess an enormous resistance to ultraviolet (UV) radiation. This is largely due to a unique DNA repair enzyme, Spore Photoproduct Lyase (SP lyase) that repairs a specific UV-induced DNA lesion, the spore photoproduct (SP), through an unprecedented radical-based mechanism. Unlike DNA photolyases, SP lyase belongs to the emerging superfamily of radical S-adenosyl-l-methionine (SAM) enzymes and uses a [4Fe-4S](1+) cluster and SAM to initiate the repair reaction. We report here the first crystal structure of this enigmatic enzyme in complex with its [4Fe-4S] cluster and its SAM cofactor, in the absence and presence of a DNA lesion, the dinucleoside SP. The high resolution structures provide fundamental insights into the active site, the DNA lesion recognition and binding which involve a ?-hairpin structure. We show that SAM and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the ?-thyminyl radical moiety, respectively. Based on structural and biochemical characterizations of mutant proteins, we substantiate the role of this cysteine in the enzymatic mechanism. Our structure reveals how SP lyase combines specific features of radical SAM and DNA repair enzymes to enable a complex radical-based repair reaction to take place.

SUBMITTER: Benjdia A

PROVIDER: S-EPMC3467042 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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

Benjdia Alhosna A Heil Korbinian K Barends Thomas R M TR Carell Thomas T Schlichting Ilme I

Nucleic acids research 20120702 18

Bacterial spores possess an enormous resistance to ultraviolet (UV) radiation. This is largely due to a unique DNA repair enzyme, Spore Photoproduct Lyase (SP lyase) that repairs a specific UV-induced DNA lesion, the spore photoproduct (SP), through an unprecedented radical-based mechanism. Unlike DNA photolyases, SP lyase belongs to the emerging superfamily of radical S-adenosyl-l-methionine (SAM) enzymes and uses a [4Fe-4S](1+) cluster and SAM to initiate the repair reaction. We report here th ...[more]

PMID: 22761404

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Project description:5-Thyminyl-5,6-dihydrothymine (also called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is repaired by a radical SAM (S-adenosylmethionine) enzyme, the spore photoproduct lyase (SPL), at the bacterial early germination phase. Our previous studies proved that SPL utilizes the 5'-dA• generated by the SAM cleavage reaction to abstract the H(6proR) atom to initiate the SP repair process. The resulting thymine allylic radical was suggested to take an H atom from an unknown protein source, most likely cysteine 141. Here we show that C141 can be readily alkylated in the native SPL by an iodoacetamide treatment, suggesting that it is accessible to the TpT radical. SP repair by the SPL C141A mutant yields TpTSO(2)(-) and TpT simultaneously from the very beginning of the reaction; no lag phase is observed for TpTSO(2)(-) formation. Should any other protein residue serve as the H donor, its presence would result in TpT being the major product at least for the first enzyme turnover. These observations provide strong evidence to support C141 as the direct H atom donor. Moreover, because of the lack of this intrinsic H donor, the C141A mutant produces TpT via an unprecedented thymine cation radical reduction (proton-coupled electron transfer) process, contrasting to the H atom transfer mechanism in the wild-type (WT) SPL reaction. The C141A mutant repairs SP at a rate that is ~3-fold slower than that of the WT enzyme. Formation of TpTSO(2)(-) and TpT exhibits a V(max) deuterium kinetic isotope effect (KIE) of 1.7 ± 0.2, which is smaller than the (D)V(max) KIE of 2.8 ± 0.3 determined for the WT SPL reaction. These findings suggest that removing the intrinsic H atom donor disturbs the rate-limiting process during enzyme catalysis. As expected, the prereduced C141A mutant supports only ~0.4 turnover, which is in sharp contrast to the >5 turnovers exhibited by the WT SPL reaction, suggesting that the enzyme catalytic cycle (SAM regeneration) is disrupted by this single mutation.

Dataset Information

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

Publications

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

Similar Datasets

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