Carbon–sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE

Abstract

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.

Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition

“…S1). The first supports Met bound as a tridentate chelate to the unique Fe through its α-amine, carboxylate, and side-chain sulfur atom, an arrangement that mirrors previously observed post-SAM cleavage structures (29)(30)(31). This is consistent with prereduction of SuiB with sodium dithionite before crystallization (8).…”

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

“…22 Despite the ability of HydE to react directly with the sulfur atom in 1,3-thiazoldine compounds, these molecules do not appear to be this enzyme’s physiological substrate because of their inability to enhance activation of [FeFe]-hydrogenase. 22 …”

Metallocofactor assembly for [FeFe]-hydrogenases

“…These crystals have been used in a crystallographic study of the reductive cleavage of SAM [31]. Some HydE sequences contain an auxiliary cluster binding site, but it is of variable constitution and can be removed by mutation without inactivating HydE, suggesting it is functionally inessential [30,32]. The structure of HydE (Figure 4a) includes a large cavity (991 Å 3 ) within the TIM barrel that is well suited to bind a second substrate (in addition to SAM).…”

“…The structure of HydE (Figure 4a) includes a large cavity (991 Å 3 ) within the TIM barrel that is well suited to bind a second substrate (in addition to SAM). Efforts to identify this substrate include in silico [24] and in vitro screening experiments [32,33]. The rate of SAM turnover to form 5ʹ-deoxyadenosine was recently used to screen a range of putative HydE second substrates, leading to the conclusion that the HydE substrate likely contains a thiol [33].…”

“…The rate of SAM turnover to form 5ʹ-deoxyadenosine was recently used to screen a range of putative HydE second substrates, leading to the conclusion that the HydE substrate likely contains a thiol [33]. Thiocyanate was identified as a fragment with significant affinity for HydE [30] and more recent studies with HydE crystals demonstrated that a small molecule, (2R,4R)-2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid, could intercept the 5ʹ-deoxyadenosyl radical intermediate, forming a new 5ʹ-C to sulfur bond in the product [32]. However, despite all of this hard won understanding, the natural substrate and products for HydE remain elusive.…”