Expanding the Chemistry of the Class C Radical SAM Methyltransferase NosN by Using an Allyl Analogue of SAM

Abstract

The radical S-adenosylmethionine (SAM) superfamily enzymes cleave SAM reductively to generate ah ighly reactive 5'-deoxyadenosyl (dAdo) radical, which initiates remarkably diverse reactions.U nlike most radical SAM enzymes,t he class Cr adical SAM methyltransferase NosN binds two SAMs in the active site,using one SAM to produce ad Ado radical and the second as am ethyl donor.H ere,w e report am echanistic investigation of NosN in which an allyl analogue of SAM (allyl-SAM) was used. We showt hat NosN cleaves allyl-SAM efficiently and the resulting dAdo radical can be captured by the olefin moieties of allyl-SAM or 5'allylthioadenosine (ATA), the latter being aderivative of allyl-SAM. Remarkably,wefound that NosN produced two distinct sets of products in the presence and absence of the methyl acceptor substrate,thus suggesting substrate-triggered production of ATAf rom allyl-SAM. We also show that NosN produces S-adenosylhomocysteine from 5'-thioadenosine and homoserine lactone.T hese results support the idea that 5'methylthioadenosine is the direct methyl donor in NosN reactions,and demonstrate great potential to modulate radical SAM enzymes for novel catalytic activities.

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] …”

“…This suggests that these analogues do not support radical generation with QCMT. Recently, it has been shown that allyl‐SAM can be a functional analogue in radical SAM enzymes [37] . However, steric restraints might impede allyl‐SAM binding to the FeS‐cluster of QCMT.…”

“…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] …”

Reductive Cleavage of Sulfoxide and Sulfone by Two Radical S-Adenosyl-l-methionine Enzymes

“…15−17 More recently, we showed that NosN is able to cleave allyl-SAM, a SAM analogue in which the methyl group is replaced by an allyl group. 18 Lin, Hoffman, and coworkers showed that several SAM analogues, in which the 3amino-3-carboxylpropyl group of SAM was modified, can be cleaved by Dph2, 19−21 an unconventional radical SAM enzyme involved in diphthamide biosynthesis. 22 However, to date, the tested SAM analogues are all confined to sulfonium salts, and it remains unclear whether the substrate scope of radical SAM enzymes can be expanded beyond sulfoniums.…”