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Radical SAM-Mediated Methylation Reactions 1,2 Available online at www.sciencedirect.com Radical SAM-mediated methylation reactions 1,2 Danica Galonic´ Fujimori A subset of enzymes that belong to the radical S- the [4Fe–4S] cluster in these proteins [3]. A subset of adenosylmethionine (SAM) superfamily is able to catalyze radical SAM enzymes has been demonstrated, or is pre- methylation reactions. Substrates of these enzymes are distinct dicted, to catalyze methylation on a diverse set of bio- from the nucleophilic substrates that undergo methylation by a molecules, some with electronic properties distinct from polar mechanism. Recently, activities of several radical SAM conventional nucleophilic methylation substrates. Akin methylating enzymes have been reconstituted in vitro and their to the profound biological roles of methyltransferses that mechanisms of catalysis investigated. The RNA modifying use polar mechanisms [4], radical SAM methylating enzymes RlmN and Cfr catalyze methylation via a methyl enzymes influence important cellular functions, such synthase mechanism. These enzymes use SAM in two distinct as regulation of translation, susceptibility to antibiotics, roles: as a source of a methyl group transferred to a conserved and maintenance of bacterial intracytoplasmic mem- 0 0 cysteine and as a source of 5 -deoxyadenosyl radical (5 -dA ). branes [5–9]. Recently, significant progress has been Hydrogen atom abstraction by this species generates a made in the reconstitution and mechanistic analysis of thiomethylene radical which adds into the RNA substrate, radical SAM methylating enzymes. This work has forming an enzyme-substrate covalent adduct. In another expanded the repertoire of known biological methylation recent study, methylation of the indole moiety of tryptophan by catalysts, as well as the scope of substrates that can the radical SAM and cobalamin-binding domain enzyme TsrM undergo enzymatic methylation. This review summar- has been reconstituted. Methylcobalamin serves as an izes these advances, and offers a perspective on out- intermediate methyl donor in TsrM, and is proposed to transfer standing questions regarding radical SAM-mediated the methyl group as a methyl radical. Interestingly, despite the methylation. presence of the radical SAM motif, no reductive cleavage of SAM has been observed in this methylation. These important Classes of radical SAM methylating enzymes reconstitutions set the stage for further studies on mechanisms Radical SAM methylating enzymes are grouped into of radical methylation. three classes based on their primary sequence [10]. Addresses The Class A subfamily is comprised of RlmN and Cfr, 1 Department of Cellular and Molecular Pharmacology, University of enzymes that methylate an adenosine in RNA (Figure 1). California, San Francisco, Genentech Hall, 600 16th Street, MC2280, These enzymes contain a radical SAM domain as their San Francisco, CA 94158-2280, USA 2 only domain. In addition to three cysteine residues Department of Pharmaceutical Chemistry, University of California, San required for the ligation of the [4Fe–4S] cluster, these Francisco, Genentech Hall, 600 16th Street, MC2280, San Francisco, CA 94158-2280, USA enzymes contain two additional conserved cysteines required for catalysis [11,12 ,13 ]. Class B enzymes Corresponding author: Fujimori, Danica Galonic´ ([email protected]) contain the radical SAM motif and a cobalamin-binding domain and modify a broad range of substrates, including heteroaromatic compounds, unactivated aliphatic car- Current Opinion in Chemical Biology 2013, 17:597–604 bons, and phosphinates [14–16,17 ,18–21,22 ,23–29]. This review comes from a themed issue on Mechanisms For example, TsrM methylates the C2 indole carbon Edited by Hung-wen Liu and Tadhg Begley of tryptophan which is ultimately incorporated into the For a complete overview see the Issue and the Editorial quinaldic acid moiety of thiostrepton [19,30 ] (Figure 1). In other examples highlighting Class B substrate diver- Available online 5th July 2013 sity, Fom3 is postulated to methylate an alcohol carbon in 1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights fosfomycin biosynthesis [14], while PoyB and PoyC are reserved. postulated to methylate a range of unactivated aliphatic http://dx.doi.org/10.1016/j.cbpa.2013.05.032 carbons in polytheonamide biosynthesis [17 ] (Figure 1). Class C enzymes methylate heteroaromatic substrates [31–35]. One such example is NosN, an enzyme that Introduction methylates the indolic acid moiety in the biosynthesis of The radical S-adenosylmethionine (SAM) enzymes com- thiopeptide antibiotic nosiheptide [31,36] (Figure 1). prise a large and functionally diverse superfamily of These enzymes contain both the radical SAM motif proteins that use radical chemistry to effect substrate and a coproporphyrinogen III oxidase HemN-like motif modifications [1]. The unifying feature of these enzymes [37]. Given the differences in domain organization, it is 0 0 is the use of 5 -deoxyadenosyl radical (5 -dA ) as a key likely that each of these three classes of enzymes use a catalytic intermediate [2]. This potent oxidant is formed distinct mechanism to catalyze methylation. Indeed, in via reductive cleavage of SAM, coordinated to an iron in recent years, different mechanisms have been shown to www.sciencedirect.com Current Opinion in Chemical Biology 2013, 17:597–604 598 Mechanisms Figure 1 O O H H O N NH O N N 2 NH NH2 H 2 N O OH N N N H C O 3 S S N N CH H O O O 3 H N N S N S N N H NH O OH O H N N N N N O HOO HO 23S rRNA H H S HN S N NH O HO O O O HN O S O NH O HN S O O HN S N N N P CH3 H C OH H NH O H 3 N S H OH N S 2 O N O C O Fosfomycin O Me HO OH HO O Thiostrepton A Nosiheptide H C H C 3 CH 3 CH O O O 3 O O 3 O O O H C H H H H H H H 3 N N N N N N N O H3C C N N N N N N N H H H H H H H CH3 O O O O O O O HN NHCH3 CH3 CH3 CH3 H2N O O HN O CH3 CH3 H C O CH O O O O O 3 O O 3 H H H H H H OH O N N N N N N NH HO N N N N N N N H H H H H H H NH O O O O O O H3C O O O OH CH3 HO −O + O NH2 O NH NHCH3 NHCH3 NHCH3 S H3C O H3C OH O O O O O O O O H H H H H H H HN N N N N N N N OH N N N N N N N N H H H H H H H H H CHN O O O O O O O O 3 O O O OH O O OH HO O NHCH3 NHCH3 NHCH3 NH2 NH2 Polytheonamides A and B Current Opinion in Chemical Biology Selected molecules that contain methyl groups or their derivatives introduced by radical SAM methylating enzymes. Groups introduced by these enzymes are shown in red. Polytheonamides A and B differ only in the absolute configuration of the sulfoxide [58]. account for methylation by Class A and Class B enzymes. RlmN [6]. Cfr modifies the C8 amidine carbon of the To date, no in vitro activity has been reconstituted for any same adenosine in 23S rRNA in pathogenic microorgan- of class C enzymes. isms [40]. The resulting modification leads to resistance to five major classes of antibiotics that target the pepti- RNA methylation by RlmN and Cfr dyltransferase center within the ribosome [7,8]. RlmN and Cfr are related bacterial enzymes that catalyze methylation of the electrophilic amidine carbons of an The activities of both RlmN and Cfr have been recon- adenosine nucleotide, A2503, in 23S ribosomal RNA stituted in vitro. Both enzymes contain a [4Fe–4S] cluster, (rRNA) [38–40]. The substrate adenosine is located at require the presence of a one electron reductant for the entrance to the nascent peptide exit tunnel of the activity, and generate both S-adenosyl-L-homocysteine 0 0 bacterial ribosome. RlmN modifies the C2 adenosine (SAH) and 5 -deoxyadenosine (5 -dA) as byproducts, con- 0 position, and the resulting modification has been shown sistent with the formation of 5 -dA and its role in hydro- to modulate interactions between the ribosome and the gen atom abstraction [41]. The mechanism of these nascent peptide, contributing to regulation and fidelity of enzymes was further investigated by the use of isotopi- translation [5,6]. More recently, it was demonstrated that cally labeled substrates [42 ] (Figure 2). In these exper- certain transfer RNAs (tRNAs) also serve as substrates for iments, deuterium incorporation into the methylated Current Opinion in Chemical Biology 2013, 17:597–604 www.sciencedirect.com Radical SAM methylation Fujimori 599 Figure 2 (a) NH2 NH2 NH2 N N 1.[methyl-d3]-SAM, N N N RlmN, SDT N HO N + N NCD2H DH2C N 2. RNA digestion O O N N R1 OH OH OH OH (b) NH2 NH2 NH N N 2 1. SAM, N N N RlmN, SDT N HO N + H C N N CH2D 3 N 2. RNA digestion O O N N D R1 OH OH OH OH (c) NH2 NH2 N 1. [methyl-d3]-SAM, N N RlmN(Cys355-CH ), SDT N 3 HO N N CH3 2. RNA digestion O N N R2 OH OH (d) NH2 NH N 2 1. SAM, N N RlmN(Cys355-CD ), SDT N 3 HO N NCD2H 2. RNA digestion O N N R2 OH OH (e) NH2 NH2 N SAM, Flv/Flx/NADPH N N N RlmN(Cys355-CD3) N DH2C N O N N R3 OH OH Current Opinion in Chemical Biology Summary of labeling experiments used to elucidate the mechanism of methylation by radical SAM methyl synthase RlmN [12 ,42 ].
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