Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5′-dAdo• “Free Radical” Is Never Free

Lysine 2,3-aminomutase (LAM) is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S–C5′ bond, which creates the highly reactive 5′-deoxyadenosyl radical (5′-dAdo•), the same radical generated by homolytic Co–C bond cleavage in B₁₂ radical enzymes. The SAM surrogate <i>S</i>-3′,4′-anhydroadenosyl-l-methionine (anSAM) can replace SAM as a cofactor in the isomerization of l-α-lysine to l-β-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of ¹³C, ²H, and ¹⁵N-labeled lysine completes the picture of how the active site of LAM from <i>Clostridium subterminale</i> SB4 “tames” the 5′-dAdo• radical, preventing it from carrying out harmful side reactions: this “free radical” in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S–C5′ bond, thereby enabling the 5′-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ∼0.6 Å toward the target and ∼1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5′ radical, with “van der Waals control” of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature

Substrate-Dependent Cleavage Site Selection by Unconventional Radical <i>S</i>‑Adenosylmethionine Enzymes in Diphthamide Biosynthesis

<i>S</i>-Adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe-4S] cluster to cleave homolytically the C_{5′,adenosine}-S bond of SAM to generate a 5′-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (<i>Ph</i>Dph2) and yeast Dph1-Dph2 instead cleave the C_γ,Met-S bond of methionine to generate a 3-amino-3-carboxylpropyl radical. We here show radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), <i>Ph</i>Dph2 cleaves the C_methyl-S bond, forming 5′-deoxy-5′-(3-aminopropylthio) adenosine (dAPTA, <b>1</b>). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]⁺ cluster. Electron-nuclear double resonance and mass spectroscopy data suggests that mechanistically one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study provides an illustration of the versatile reactivity of Fe-S clusters

Organometallic Complex Formed by an Unconventional Radical <i>S</i>‑Adenosylmethionine Enzyme

<i>Pyrococcus horikoshii</i> Dph2 (<i>Ph</i>Dph2) is an unusual radical <i>S</i>-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAM_CA), in which the ACP group of SAM is replaced with a 3-carboxy­allyl group. SAM_CA is cleaved by <i>Ph</i>Dph2, yielding a paramagnetic (<i>S</i> = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron–nuclear double resonance (ENDOR) measurements with ¹³C and ²H isotopically labeled SAM_CA support a π-complex between the CC double bond of α-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM-related [4Fe-4S]⁺ cluster forming an organometallic complex with an alkene, shedding additional light on the mechanism of <i>Ph</i>Dph2 and expanding our current notions for the reactivity of [4Fe-4S] clusters in radical SAM enzymes

Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme

Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B₁₂ enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na⁺ as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by ²³Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M⁺ ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron–sulfur cluster. ENDOR studies of the PFL-AE/[¹³C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little–no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li⁺ to Cs⁺, PFL-AE activity sharply maximizes at K⁺, with NH₄⁺ closely matching the efficacy of K⁺. PFL-AE is thus a type I M⁺-activated enzyme whose M⁺ controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron–sulfur cluster