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Radical SAM Enzyme Spore Photoproduct Lyase: Properties of the ? Organometallic Intermediate and Identification of Stable Protein Radicals Formed during Substrate-Free Turnover.

ABSTRACT: Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]¹⁺ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S-C5' bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate ? in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5' of the 5'-deoxyadenosyl radical (5'-dAdo^•). During catalysis, homolytic cleavage of the Fe-C5' bond liberates 5'-dAdo^• for reaction with substrate, but here, we use ? formation without substrate to determine the thermal stability of ?. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms ? within ?15 ms after mixing. By monitoring the decay of ? through rapid freeze-quench trapping at progressively longer times we find an ambient temperature decay time of the ? Fe-C5' bond of ? ? 5-6 s, likely shortened by enzymatic activation as is the case with the Co-C5' bond of B₁₂. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5'-dAdo^• radical liberated by ?. In the absence of substrate, ? undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val^•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile^• or Cys^• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.

SUBMITTER: Pagnier A

PROVIDER: S-EPMC7730139 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Radical SAM Enzyme Spore Photoproduct Lyase: Properties of the Ω Organometallic Intermediate and Identification of Stable Protein Radicals Formed during Substrate-Free Turnover.

Pagnier Adrien A Yang Hao H Jodts Richard J RJ James Christopher D CD Shepard Eric M EM Impano Stella S Broderick William E WE Hoffman Brian M BM Broderick Joan B JB

Journal of the American Chemical Society 20201015 43

Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]1+ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S-C5' bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5' of the 5'-deoxyadenosyl radical (5'-dAdo•). During catalys ...[more]

PMID: 32966073

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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

Radical SAM Enzyme Spore Photoproduct Lyase: Properties of the ? Organometallic Intermediate and Identification of Stable Protein Radicals Formed during Substrate-Free Turnover.

Publications

Radical SAM Enzyme Spore Photoproduct Lyase: Properties of the Ω Organometallic Intermediate and Identification of Stable Protein Radicals Formed during Substrate-Free Turnover.

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