Bacterial spores are remarkable in their resistance to chemical and physical stresses, including exposure to UV radiation. The unusual UV resistance of bacterial spores is a result of the unique photochemistry of spore DNA coupled with the efficient repair of accumulated damage. Exposure of bacterial spores to UV radiation results in the formation of a methylene-bridged thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP), as the primary photoproduct. 1-3 SP accumulates in UV-irradiated spores, however it is rapidly repaired upon germination, thus giving rise to the extraordinary UV resistance of bacterial spores. 4,5 The repair of SP is catalyzed by the enzyme spore photoproduct lyase (SPL), and involves the direct reversal of SP to two thymines without base excision (Scheme 1).SPL is a member of the radical AdoMet superfamily, and utilizes a [4Fe-4S] cluster and Sadenosylmethionine (AdoMet) as essential cofactors in SP repair. 6-9 We have previously shown that SP repair is initiated by abstraction of H• from C6 of SP by an AdoMet-derived 5′-deoxyadenosyl radical; 10,11 this H-atom abstraction is thought to initiate a radical-mediated β-scission of the C5-C bridge bond in the photoproduct, as originally proposed by Mehl and Begley. 12 While two distinct diastereomers of SP (5R or 5S, Fig 1) could in principle be formed upon UV irradiation of bacterial spores, only the 5R configuration is possible for SP formed from adjacent thymines in double helical DNA, due to the constraints imposed by the DNA structure. 13 The 5S configuration, therefore, is possible only in less well-defined DNA structures or as an interstrand crosslink. It was thus quite surprising when two recent reports concluded that SPL repairs only the 5S, and not the 5R, isomer of a synthetic SP substrate. 14,15 We report here results from HPLC and MS analysis of in vitro enzymatic assays on stereochemicallydefined synthetic SP substrates demonstrating that SPL specifically repairs only the 5R isomer of SP. This stereospecific repair of 5R-SP by SPL is consistent with the longstanding hypothesis that SP is a result of UV-induced dimerization of adjacent thymines in double-helical DNA.SPL was cloned from Clostridium acetobutylicum, overexpressed in Escherichia coli, and purified using a method similar to published procedures. 11 The enzyme contained 2.9 (± 0.2) Fe per SPL, and had UV-visible and EPR spectroscopic properties characteristic of an ironsulfur enzyme. The 5R and 5S diastereomers of protected (N-SEM, O-TES, and O-TBDMS) SP were synthesized using modifications of published procedures, 13,15 and were subsequently deprotected (Supporting Information). The structures of the fully protected, the di-SEM protected, and fully deprotected dinucleoside spore products were confirmed by 1 H and 13 C NMR techniques, and NOESY and ROESY were used to assign the stereochemistry at C-5 (S.I.). In order to remove any potential ambiguity associated with the assignment of stereochemistry at C-5 in the open dinucleoside forms of SP, the...
Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S–C5′ bond, which creates the highly reactive 5′-deoxyadenosyl radical (5′-dAdo•), the same radical generated by homolytic Co–C bond cleavage in B12 radical enzymes. The SAM surrogate S-3′,4′-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-β-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of 13C, 2H, and 15N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 “tames” the 5′-dAdo• radical, preventing it from carrying out harmful side reactions: this “free radical” in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S–C5′ bond, thereby enabling the 5′-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ~0.6 Å toward the target and ~1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5′ radical, with “van der Waals control” of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature.
We report the preparation of [5,10,15,20-tetraphenyl-2,3,7,8,12,13,17,18-octakis(phenylethynyl)porphinato] complexes of Ni(II), H(2), Zn(II), Mg(II), and Cu(II), as well as select trimethylsilanylethynyl derivatives. The X-ray structures of the octakis(phenylethynyl) compounds show systematic deviations from planarity (Ni(II), 0.2851 A; Zn(II), 0.0304 A) as a function of the central cation. These geometric distortions are reflected in bathochromic shifts of the Soret and Q bands (Ni(II), 497, 604, and 650 nm; Mg(II), 515, 595, 642, and 705 nm) which loosely correlate with increasing planarity of the structure. Similarly, vibrational modes involving the octasubstituted porphyrin core exhibit shifts to lower frequency as a function of increasing planarity in the solution-state resonance Raman spectra (lambda(exc) = 501.7 nm) of these compounds. Analogous trends are also observed in their solid-state electronic and resonance Raman spectra, indicating that the structural distortions within the octakis(phenylethynyl) series are preserved in solution. Comparison of the saddle distortion of the octasubstituted Ni(II) compound with the ruffle/saddle distortions of the pentakis and hexakis Ni(II) derivatives reveals some influence of asymmetric peripheryl substitution on geometric structure. These Ni(II) derivatives also exhibit systematic red shifts in their electronic spectra as a function of the number of conjugated alkyne units ( approximately 13 nm/alkyne), revealing participation of the enediyne units in the electronic ground and excited states. The solid-state Bergman cyclization temperatures of the phenylethynyl compounds vary markedly as a function of planarity, and correlate loosely with alkyne termini separation (Ni(PA)(8), 4.00 A, 281 degrees C; MgP(PA)(8), 3.77 A, 244 degrees C). In solution, both thermal and photochemical activation of the free-base octakis(phenylethynyl) compound lead to formal reduction of the porphyrin backbone via H-atom addition at opposing meso-positions. Generation of a common product suggests that both thermal and photochemical pathways to Bergman cyclization in solution contain significant activation barriers to formation of the 1,4-phenyl diradical intermediate, and under these solution conditions, alternate reaction channels are more thermodynamically favorable.
Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a [3Fe-4S] + cluster accounting for 3-4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe-4S] + cluster is observed that accounts for 34-45% of total iron. Addition of S-adenosyl-methionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-Lmethionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 ± 0.6 nmol min −1 mg −1 , whereas no repair was observed for the 5S diastereomer.
Novel indoline ribonucleosides with the alpha-N-glycoside configuration are synthesized with very high regioselectivity in 90-96%yield, using TMS protected indolines and 2,3-O-(1-methylethylidene)-5-O-(triphenylmethyl)-alpha/beta-D-ribofuranose. The structures of these ribonucleosides were elucidated with X-ray crystallography as well as 2D (NOESY, COSY, and HMQC) NMR spectroscopy.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.