A Radically Different Mechanism for
            <i>S</i>
            -Adenosylmethionine–Dependent Methyltransferases

Grove, Tyler L.; Benner, Jack S.; Radle, Matthew I.; Ahlum, Jessica H.; Landgraf, Bradley J.; Krebs, Carsten; Squire, Jeremy A.

doi:10.1126/science.1200877

Cited by 210 publications

(351 citation statements)

References 38 publications

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“…1, step 4). With few exceptions (36,37), reduction of the AdoMet cluster during catalysis has only been proposed in systems that, unlike anSMEcpe, use AdoMet catalytically. Assuming that Aux I is the electron acceptor, for a subsequent turnover, it would need to be reoxidized.…”

Section: Discussionmentioning

confidence: 99%

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Goldman¹,

Grove²,

Sites³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Arylsulfatases require a maturating enzyme to perform a co-or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S-adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6-1.8 Å resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidylsubstrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteinerich C-terminal domain, termed SPASM domain, that contains homology to ∼1,400 other unique AdoMet radical enzymes proposed to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.iron-sulfur cluster fold | radical SAM dehydrogenase

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Section: Discussionmentioning

confidence: 99%

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Goldman¹,

Grove²,

Sites³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

112

197

View full text Add to dashboard Cite

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“…SAM is involved in a wide variety of metabolic roles including methylation of many small molecules and of macromolecules (DNA, RNA and others), synthesis of spermidine, and formation of the catalytic group of a diverse group of radical SAM enzymes (Greene, 1996;Grove et al, 2011). This versatility depends on the sulfur atom of its chiral sulfonium group, which carries three activated substituents: methyl, aminopropylcarboxylate and 5-deoxyadenosine groups each involved in different sorts of reactions.…”

Section: Introductionmentioning

confidence: 99%

Cell division, one-carbon metabolism and methionine synthesis in a metK-deficient Escherichia coli mutant, and a role for MmuM

2013

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An E. coli K-12 mutant deficient in S-adenosylmethionine (SAM) synthesis, i.e DmetK, but expressing a rickettsial SAM transporter, can grow in glucose minimal medium if provided with both SAM and methionine. It uses the externally provided (R)-enantiomer of SAM as methyl donor to produce most but not all of its methionine, by methylation of homocysteine catalysed by homocysteine methyltransferase (MmuM). The DmetK cells are also altered in growth and are twice as long as those of the parent strain. When starved of SAM, the mutant makes a small proportion of very long cells suggesting a role of SAM and of methylation in the onset of crosswall formation.

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“…Lack of methylation of A2503 has been reported to produce a subtle yet significant effect on cell fitness and to increase linezolid resistance (Toh et al 2008;Gao et al 2010;Lamarre et al 2011). RlmN and its evolutionarily related resistance enzyme Cfr belong to the radical S-adenosyl-L-methionine (SAM) enzyme superfamily, and both use protein-free rRNA as a substrate (Atta et al 2010;Kaminska et al 2010;Yan et al 2010;Grove et al 2011;Yan and Fujimori 2011).…”

Section: Introductionmentioning

confidence: 99%

The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

Benı́tez-Páez

Villarroya

Armengod

2012

RNA

103

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Modifying RNA enzymes are highly specific for substrate-rRNA or tRNA-and the target position. In Escherichia coli, there are very few multisite acting enzymes, and only one rRNA/tRNA dual-specificity enzyme, pseudouridine synthase RluA, has been identified to date. Among the tRNA-modifying enzymes, the methyltransferase responsible for the m 2 A synthesis at purine 37 in a tRNA set still remains unknown. m 2 A is also present at position 2503 in the peptidyl transferase center of 23S RNA, where it is introduced by RlmN, a radical S-adenosyl-L-methionine (SAM) enzyme. Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The DrlmN mutant lacks m 2 A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. Moreover, RlmN performs m 2 A 37 synthesis in vitro using a tRNA chimera as a substrate. This chimera has also proved useful to characterize some tRNA identity determinants for RlmN and other tRNA modification enzymes. Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m 2 A 37 from tRNA is expected to produce a hyperaccurate phenotype, we believe that the error-prone phenotype exhibited by the DrlmN mutant is due to loss of m 2 A from 23S rRNA and, accordingly, that the m 2 A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center.

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A Radically Different Mechanism for S -Adenosylmethionine–Dependent Methyltransferases

Cited by 210 publications

References 38 publications

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Cell division, one-carbon metabolism and methionine synthesis in a metK-deficient Escherichia coli mutant, and a role for MmuM

The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

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