Studies on the mechanism of lysine 2,3-aminomutase

Aberhart, D. John

doi:10.1039/p19880000343

Cited by 12 publications

(15 citation statements)

References 4 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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“…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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“…They proposed that the dAdoH moiety of SAM played a role analogous to that of the dAdoH moiety of adenosylcobalamin in B 12 -dependent rearrangements. Although there were contradictory reports subsequently published, 202 these initial results from the Frey lab have been substantiated by numerous additional studies.…”

Section: Radical Sam Mutasesmentioning

confidence: 98%