Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis

Abstract

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5′-deoxyadenosyl radical (5′-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe–C5′ bond between 5′-dAdo· and the [4Fe–4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5′-dAdo· intermediate. Liberation of a reactive 5′-dAdo· intermediate via homolytic metal–carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.

Introduction of d‐Amino Acids in Minimalistic Peptide Substrates by an S‐Adenosyl‐l‐Methionine Radical Epimerase

“…These observed differences may be inherent enzyme properties or result from specific reaction conditions.N otably,O spD and PoyD share early catalytic steps with other AdoMet radical superfamily members.F ollowing photoreduction in rapidquench experiments,E PR spectra were consistent with formation of organometallic intermediate W with an Fe-C5' bond between dAdoC and the [4Fe-4S] cluster. [25] Homolytic cleavage of the organometallic bond liberates dAdoC,w hich abstracts the a-hydrogen of the target amino acid, as shown here for OspD.T he intermediate catalytic steps remain to be elucidated, but may involve aconserved epimerase cysteineas the solvent-exchangeable H-donor to quench the substrate radical, consistent with deuterium labeling of epimerized sites in D 2 O. [11] Further to these mechanistic insights,t he bioengineering potential of an AdoMet proteusin epimerase was explored to introduce stability-promoting d-amino acid residues into av ariety of peptides.L eader-independent substrates are so far limited to peptides related to the native precursor cores.…”

Mechanism of Diol Dehydration by a Promiscuous Radical‐SAM Enzyme Homologue of the Antiviral Enzyme Viperin (RSAD2)

“…Human viperin is shown to be a member of the radical‐SAM superfamily of enzymes, which catalyse the transformation of a substrate by using a common catalytic step (Figure A): a highly conserved [4 Fe−4 S] cluster, which is coordinated to three cysteine residues of a conserved CxxxCxxC motif, cleaves SAM in a one‐electron reduction step to generate a 5′‐deoxyadenosyl radical (5′‐dAdo radical) intermediate . The 5′‐dAdo radical has recently been trapped and characterised, and spectroscopic studies have provided evidence for the formation of an organometallic intermediate with a Fe‐[5′‐C]‐deoxyadenosyl bond (Figure A) . The organometallic or the 5′‐dAdo radical intermediate initiates catalytic transformation by abstracting a hydrogen atom from a substrate, releasing 5′‐deoxyadenosine (5′‐dA), and generating a substrate–radical intermediate that undergoes complex rearrangement reactions to form a product .…”

The hidden enzymology of bacterial natural product biosynthesis

“…They are known to catalyse a diverse array of chemically challenging transformations, many of which involve the selective activation of inert C-H bonds. Each transformation is initiated by a common mechanism of radical generation via an organometallic Ω intermediate 144 , in which a reduced [4Fe-4S] cluster cofactor homolytically cleaves the SAM 5′-C-S bond to generate Met and a highly reactive 5′-deoxyadenosyl radical (dAdo•). This radical usually abstracts a substrate hydrogen, setting the stage for an incredibly diverse range of transformations that convert resulting substrate radicals into products.…”