Mechanistic Studies of the Spore Photoproduct Lyase via a Single Cysteine Mutation

Abstract

5-thyminyl-5,6-dihydrothymine (also called spore photoproduct or SP) is the exclusive DNA photo-damage 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 SAM cleavage reaction to abstract the H6proR 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 the cysteine 141. Here we show that C141 can be readily alkylated in the native SPL by iodoacetamide treatment, suggesting that it is accessible to the TpT radical. SP repair by the SPL C141A mutant yields TpTSO2− and TpT simultaneously from the very beginning of the reaction; no lag phase is observed for the TpTSO2− formation. Should any other protein residue serve as the H donor, its presence would result in TpT as 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, due to 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 WT SPL reaction. The C141A mutant repairs SP at a rate which is ~3-fold slower than the WT enzyme. Formation of TpTSO2− and TpT exhibit a Vmax deuterium kinetic isotope effect (KIE) of 1.7 ± 0.2 respectively, which is smaller than the DVmax KIE of 2.8 ± 0.3 determined in the WT SPL reaction. These findings suggest that removing the intrinsic H atom donor disturbs the rate-limiting process in the enzyme catalysis. As expected, the pre-reduced C141A mutant only supports ~ 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.

Does Viperin Function as a Radical S-Adenosyl-l-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity?

“…IPP and GPP were purchased from Isoprenoids. [4][5][6][7][8][9][10][11][12][13][14] C]IPP was purchased from PerkinElmer Life Sciences. HEK293T cell line was obtained from ATCC.…”

“…For example, radical SAM-dependent enzymes participate in the biosynthesis of herbicides, antibiotics, vitamins, co-factors such as biotin and thiamin, and various other natural products (2,(5)(6)(7)(8). They also function in the modification of ribosomal and transfer RNAs (9,10) and DNA repair (11) and in the post-translational modification of peptides and proteins (12)(13)(14). Sequence analyses indicate that there are potentially thousands of members of the radical SAM superfamily, but to date, relatively few of these enzymes have been isolated and characterized (15,16).…”

“…Ado ⅐ subsequently abstracts a hydrogen atom from the substrate, thereby forming 5Ј-deoxyadenosine (5Ј-dA) and a substrate radical that undergoes further chemical reaction. Most radical SAM enzymes consume SAM as a co-substrate during catalysis; however, in a few enzymes, including lysine 2,3-aminomutase (18), spore photoproduct lyase (11), and DesII (7), Ado ⅐ formation is reversible, and SAM is regenerated as a co-factor.…”

The radical SAM enzyme spore photoproduct lyase employs a tyrosyl radical for DNA repair

“…6 The SPL catalyzes the direct repair of the SP-lesion, utilizing a radical mediated mechanism (Fig. 1A) 13 showed that the allyl reducing moiety is a conserved cysteine. A recent crystal structure of the SPL from Geobacillus thermodenitrificans 14 in complex with the lesion 15 ( Fig.…”

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.…”