Lysine 2,3-aminomutase: role of S-adenosyl-L-methionine in the mechanism. Demonstration of tritium transfer from (2RS,3RS)-[3-3H]lysine to S-adenosyl-L-methionine

Kilgore, James L.; Aberhart, D. John

doi:10.1039/p19910000079

Cited by 24 publications

(14 citation statements)

References 19 publications

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“…Previous efforts to establish the SAM regeneration were conducted via a tritium labeling experiment in SPL and lysine-2,3-aminomutase (LAM) (10, 20, 37). In both cases, the percentage of label transfer from the substrate into SAM was suggested to be very low (39, 96, 97). Recent proposals by Eguchi in the studies of BtrN (98) and by Liu in the studies of DesII (27), both of which are radical SAM enzymes, suggested that SAM cleavage, as well as the subsequent H atom abstraction by the 5'-dA• from enzyme substrates, may be reversible.…”

Section: Discussionmentioning

confidence: 97%

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

et al. 2012

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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.

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Section: Discussionmentioning

confidence: 97%

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

et al. 2012

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“…11 Interestingly in LAM where SAM also appears regenerated after each catalytic cycle, no deuterium transfer from lysine to SAM was observed either, 45 although a more sensitive tritium transfer experiment revealed ~ 1 - 6% of tritium incorporation from the lysine substrate into SAM when large excess of SAM was adopted. 46 …”

Section: Resultsmentioning

confidence: 99%

“…Any deuterium incorporated into SAM is expected to be retained in the resulting MTA, which was analyzed by LC-MS using the protocol developed previously in mechanistic studies of lysine 2,3-aminomutase (LAM). 45,46 …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2011

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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.

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“…Further support for this role for SAM in the LAM-catalyzed reaction was provided by utilizing S-[5′- 3 H]-adenosylmethionine in the presence of excess LAM, which resulted in all of the tritium ending up in lysine or β-lysine. 203 The tritium transfer from the 5′-position of SAM into the reactant/product of LAM, together with further label transfer experiments utilizing [3,3- 2 H 2 ]-lysine 203 or [3- 3 H]-lysine, 204 provided evidence that SAM served as a precursor of a dAdo • during LAM catalysis, and that this radical intermediate mediated hydrogen transfer from C3 to C2 of lysine. Using SAM labeled with 14 C at either the carboxyl carbon of the methionine moiety or the 8-position of the adenine ring, Moss and Frey demonstrated the conversion of SAM to methionine and dAdoH during LAM catalysis.…”

Section: Radical Sam Mutasesmentioning

confidence: 99%

RadicalS-Adenosylmethionine Enzymes

et al. 2014

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Lysine 2,3-aminomutase: role of S-adenosyl-L-methionine in the mechanism. Demonstration of tritium transfer from (2RS,3RS)-[3-3H]lysine to S-adenosyl-L-methionine

Cited by 24 publications

References 19 publications

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

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

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

RadicalS-Adenosylmethionine Enzymes

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