- Radical SAM Enzyme HydE Generates Adenosylated Fe(I) Intermediates En Route to the [FeFe]-Hydrogenase Catalytic H-Cluster
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The H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S]H-subcluster linked by a cysteinyl bridge to a unique organometallic [2Fe]H-subcluster assigned as the site of interconversion between protons and molecular hydrogen. This [2Fe]H-subcluster is assembled by a set of Fe-S maturase enzymes HydG, HydE and HydF. Here we show that the HydG product [FeII(Cys)(CO)2(CN)] synthon is the substrate of the radical SAM enzyme HydE, with the generated 5′-deoxyadenosyl radical attacking the cysteine S to form a C5′-S bond concomitant with reduction of the central low-spin Fe(II) to the Fe(I) oxidation state. This leads to the cleavage of the cysteine C3-S bond, producing a mononuclear [FeI(CO)2(CN)S] species that serves as the precursor to the dinuclear Fe(I)Fe(I) center of the [2Fe]H-subcluster. This work unveils the role played by HydE in the enzymatic assembly of the H-cluster and expands the scope of radical SAM enzyme chemistry.
- Begley, Tadhg P.,Britt, R. David,Joshi, Sumedh,Pattenaude, Scott A.,Rauchfuss, Thomas B.,Tao, Lizhi
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- Carbon-sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE
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Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5′-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.
- Rohac, Roman,Amara, Patricia,Benjdia, Alhosna,Martin, Lydie,Ruffié, Pauline,Favier, Adrien,Berteau, Olivier,Mouesca, Jean-Marie,Fontecilla-Camps, Juan C.,Nicolet, Yvain
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p. 491 - 500
(2016/05/09)
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- S-homoadenosyl-L-cysteine and S-homoadenosyl-L-homocysteine. Synthesis and binding studies of non-hydrolyzed substrate analogues with S-adenosyl-L-homocysteine hydrolase
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Treatment of homoadenosine [9-(5-deoxy-β-D-ribo-hexofuranosyl)adenine] with thionyl chloride and pyridine in acetonitrile gave 6′-chloro-6′-deoxyhomoadenosine, which underwent nucleophilic displacement with L-cysteine or L-homocysteine to give homologated analogues of S-adenosyl-L-homocysteine. Each amino acid in aqueous sodium hydroxide at 60 °C gave excellent conversion from the chloronucleoside, and adsorption on Amberlite XAD-4 resin provided more convenient isolation than prior methods. Weak binding of these non-hydrolyzed analogues to S-adenosyl-L-homocysteine hydrolase was observed.
- Miles, Robert W.,Nielsen, Lars P. C.,Ewing, Gregory J.,Yin, Daniel,Borchardt, Ronald T.,Robins, Morris J.
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p. 8258 - 8260
(2007/10/03)
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- AN IMPROVED SYNTHESIS OF S-ADENOSYL-L-HOMOCYSTEINE AND RELATED COMPOUNDS
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5'-Chloro-5'-deoxy-2',3'-O-isopropylideneadenosine reacts with disodium salts of L-homocysteine, L-cysteine or 3-mercaptopropanoic acid in liquid ammonia to afford 2',3'-O-isopropylidene derivatives which are easily desalted by chromatography on octadecyl-silica column.On acid treatment, the high purity preparations of S-adenosyl-L-homocysteine, S-adenosyl-L-cysteine, and 5'-carboxyethylthio-5'-deoxyadenosine are obtained in respectable yield.
- Holy, Antonin,Rosenberg, Ivan
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p. 1514 - 1518
(2007/10/02)
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