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

Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-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.

Radical SAM catalysis via an organometallic intermediate with an Fe-[5'-C]-deoxyadenosyl bond.

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5'-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and (13)C, (57)Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5' carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes.

Radical SAM enzymes in methylation and methylthiolation.

Radical S-adenosyl-l-methionine (SAM) enzymes are a large and diverse superfamily with functions ranging from enzyme activation through a single H atom abstraction to complex organic and metal cofactor synthesis involving a series of steps. Though these enzymes carry out a variety of functions, they share common structural and mechanistic characteristics. All of them contain a site-differentiated [4Fe-4S] cluster, ligated by a CX(3)CX(2)C or similar motif, which binds SAM at the unique iron. The [4Fe-4S](1+) state of the cluster reductively cleaves SAM to produce a 5'-deoxyadenosyl radical, which serves to initiate the diverse reactions catalyzed by these enzymes. Recent highlights in the understanding of radical SAM enzymes will be presented, with a particular emphasis on enzymes catalyzing methylation and methythiolation reactions.