S‐Adenosyl‐l‐ethionine is a Catalytically Competent Analog of S‐Adenosyl‐l‐methionine (SAM) in the Radical SAM Enzyme HydG

Abstract

Radical S‐adenosyl‐l‐methionine (SAM) enzymes initiate biological radical reactions with the 5′‐deoxyadenosyl radical (5′‐dAdo.). A [4Fe‐4S]+ cluster reductively cleaves SAM to form the Ω organometallic intermediate in which the 5′‐deoxyadenosyl moiety is directly bound to the unique iron of the [4Fe‐4S] cluster, with subsequent liberation of 5′‐dAdo.. We present synthesis of the SAM analog S‐adenosyl‐l‐ethionine (SAE) and show SAE is a mechanistically equivalent SAM‐alternative for HydG, both supporting enzymatic turnover of substrate tyrosine and forming the organometallic intermediate Ω. Photolysis of SAE‐bound HydG forms an ethyl radical trapped in the active site. The ethyl radical withstands prolonged storage at 77 K and its EPR signal is only partially lost upon annealing at 100 K, making it significantly less reactive than the methyl radical formed by SAM photolysis. Upon annealing above 77 K, the ethyl radical adds to the [4Fe‐4S]2+ cluster, generating an ethyl‐[4Fe‐4S]3+ organometallic species termed ΩE.

Mechanism of Radical Initiation in the Radical SAM Enzyme Superfamily

“…The importance of active site forces in controlling radical intermediates is further demonstrated by a study involving the SAM analog S-adenosylethionine (SAE) in which the methyl group of SAM is replaced by an ethyl group (43). This change does not alter the observed regioselectivity of photocleavage: Photolysis of PFL-AE-SAE, like PFL-AE-SAM, gives rise to a 5 ′ -dAdo r trapped in the active site, while photolysis of HydG-SAE results in homolysis of the Sethyl bond to produce an ethyl radical ( r CH 2 CH 3 ) trapped in the active site (43,44). Compared to the methyl radical produced from HydG-SAM photolysis, the ethyl radical is more constrained in the active site, and like the larger 5 ′ -dAdo r in PFL-AE, the ethyl radical is indefinitely stable at 77 K (44).…”

“…Compared to the methyl radical produced from HydG-SAM photolysis, the ethyl radical is more constrained in the active site, and like the larger 5 ′ -dAdo r in PFL-AE, the ethyl radical is indefinitely stable at 77 K (44). The ethyl radical trapped in the HydG active site anneals to an organometallic species denoted E in which the ethyl group is covalently bound to the unique iron of the [4Fe-4S] cluster (Figure 10) (43).…”

Biomimetic S‐Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes**

“…In addition, the alkylcobalamin must be able to react with the substrate radical. The ability to generate the DOA⋅ radical from SAM analogues has been previously demonstrated for Se ‐adenosylselenomethionine ( Se SAM), SAE, allyl‐SAM, nucleobase analogues, and the sulfoxide derivative of SAH [36–38,84,85] . Alkylcobalamins have been described in some studies, including enzyme‐catalysed C−C bond formation [86,87] …”

“…Particularly in drug synthesis the ever‐expanding chemical space of radical SAM enzymes is of interest for challenging reactions, e. g. stereo‐ and regioselective methylation of sp 3 ‐carbons. In addition to the possibility of broadening the diversity of radical SAM MT products by transferring alternative alkyl chains, this might be interesting in terms of using SAM analogues for mechanistic studies as has been shown before [36,37,84,85] . Together with the increasing number of available enzyme structures, it might be possible to design enzyme variants with a broader or altered substrate range, as has been shown for MATs and conventional MTs [88–91] …”

“…MTs have been used together with alkyl analogues of SAM to transfer bulkier residues [33–35] . Radical SAM enzymes have been shown to support the cleavage of S ‐adenosylethionine (SAE) and S ‐adenosylallylhomocysteine (allyl‐SAM) as well as the transfer of various nucleosides to substrate analogues [36–38] …”