Probing the Reaction Mechanism of Spore Photoproduct Lyase (SPL) via Diastereoselectively Labeled Dinucleotide SP TpT Substrates

Abstract

5-thyminyl-5,6-dihydrothymine (commonly called spore photoproduct or SP) is the exclusive DNA photo-damage product in bacterial endospores. It is generated in the bacterial sporulation phase and repaired by a radical SAM enzyme, spore photoproduct lyase (SPL), at the early germination phase. SPL utilizes a special [4Fe-4S] cluster to reductively cleave S-adenosylmethionine (SAM) to generate a reactive 5′-dA radical. The 5′-dA radical is proposed to abstract one of the two H atoms at the C6 carbon of SP to initiate the repair process. Via organic synthesis and DNA photochemistry, we selectively labeled the 6-HproS or 6-HproR position with a deuterium in a dinucleotide SP TpT substrate. Monitoring the deuterium migration in enzyme catalysis (employing Bacillus subtilis SPL) revealed that it is the 6-HproR atom of SP that is abstracted by the 5′-dA radical. Surprisingly, the abstracted deuterium was not returned to the resulting TpT after enzymatic catalysis, an H atom from the aqueous buffer was incorporated into TpT instead. This result questions the currently hypothesized SPL mechanism which excludes the involvement of protein residue(s) in SPL reaction, suggesting that some protein residue(s), which is capable of exchanging a proton with the aqueous buffer, is involved in the enzyme catalysis. Moreover, evidence has been obtained for a possible SAM regeneration after each catalytic cycle; however, such a regeneration process is more complex than currently thought, with one or even more protein residues involved as well. These observations have enabled us to propose a modified reaction mechanism for this intriguing DNA repair enzyme.

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

“…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. SPL activity using dinucleotide SP TpT as the substrate was reported to be ϳ0.3 min Ϫ1 by different groups (72,75,77,80), suggesting that the enzymes used were similarly active. Unpublished results from our laboratory, 3 however, found SPL to repair SP in a 20-nucleotide duplex prepared via DNA synthesis (81) at only 0.08 Ϯ 0.01 min Ϫ1 .…”

“…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.…”

“…To reveal which hydrogen atom is abstracted during SPL catalysis, Lin et al (71) utilized deuterium-labeled dinucleotide TpTs to generate two SP species via photoreaction and proved that a hydrogen atom from the 3Ј-CH 3 is transferred to the H 6proS of SP. Using SP with either the H 6proR or the H 6proS position labeled by a deuterium, Yang et al (72) proved that it is the H 6proR that is abstracted by the 5Ј-dA ⅐ in SP repair (Fig. 2).…”

“…Thus, no radioactivity loss in DNA was observed, leading to the hypothesis that SPL directly reverts SP. If the researchers chose to label thymine C6, the label at the resulting H 6proR of SP would leak into medium after SP repair (72), potentially complicating the SPL mechanistic elucidation.…”

“…A later in vitro study by Chandor-Proust et al (74) found that the thymine allylic radical was quenched by the dithionite supplemented as a reductant to form a T-SO 2 Ϫ adduct, leading to a hypothesis that Cys-141 may be the hydrogen atom donor. Yang et al (72) found that the hydrogen donor is able to exchange protons with the aqueous solution. They showed that Cys-141 is solvent-accessible via the iodoacetamide-labeling assay; their enzyme kinetics data further indicate that the Cys-141 in Bacillus subtilis (Bs) SPL is likely the intrinsic hydrogen donor to the thymine radical ( Fig.…”