Structural Basis for Methyl Transfer by a Radical SAM Enzyme

Boal, Amie K.; Grove, Tyler L.; McLaughlin, Martin I.; Yennawar, Neela H.; Booker, Squire J.; Rosenzweig, Amy C.

doi:10.1126/science.1205358

Cited by 158 publications

(197 citation statements)

References 47 publications

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“…Initial work on these enzymes showed that SAM acts both as a source of Ado-CH 2 · and as a methyl group donor in the methylation reactions (36). Later, extensive mechanistic and crystallographic studies revealed that RlmN catalyzes an initial S N 2 nucleophilic substitution reaction to methylate a cysteine residue on the enzyme and uses subsequent radical SAM-dependent chemistry to generate a methyl group on the rRNA substrate (37)(38)(39). In the latter reaction, the methyl group that is appended to the adenosine residue retains only two hydrogens from the original SAM-derived methyl group.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Allen

White

2014

J Bacteriol

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Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-L-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX 3 CX 2 C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.

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Section: Resultsmentioning

confidence: 99%

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Allen

White

2014

J Bacteriol

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“…S2). Moreover, formation of the same reaction byproducts by NifB proteins as those by radical SAM RNA methyltransferases RlmN and Cfr (19,20) points to a similarity between NifB and these two well-characterized members of a larger subset of radical SAM enzymes that catalyze methylation reactions using SAM or other methyl donor molecules as cosubstrates (see Discussion). Interestingly, NifB Ma and NifB Mt appeared to be more efficient than NifB Av in cleaving SAM into SAH and 5′-dAH, as a substantial amount of SAM was left uncleaved when it was incubated with NifEN-B (Fig.…”

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confidence: 99%

“…5B, 2). This observation led to an important question of whether the residual radiolabel on NifB Mt originated from transfer of the 14 C-labeled methyl group to a certain protein residue as an intermediary step of carbide insertion, which would suggest a reaction pathway somewhat analogous to the one used by RlmN and Cfr to facilitate methylation of an inert C-H bond (19,20). To address this question, NifB Ma and NifB Mt were prepared in the absence and presence of SAM and subsequently analyzed for posttranslational modification (PTM).…”

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confidence: 99%

Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens

Fay

Wiig

Lee

et al. 2015

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

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Nitrogenase biosynthesis protein NifB catalyzes the radical S-adenosyl-L-methionine (SAM)-dependent insertion of carbide into the M cluster, the cofactor of the molybdenum nitrogenase from Azotobacter vinelandii. Here, we report the identification and characterization of two naturally "truncated" homologs of NifB from Methanosarcina acetivorans (NifB Ma ) and Methanobacterium thermoautotrophicum (NifB Mt ), which contain a SAM-binding domain at the N terminus but lack a domain toward the C terminus that shares homology with NifX, an accessory protein in M cluster biosynthesis. NifB Ma and NifB Mt are monomeric proteins containing a SAM-binding [Fe 4 S 4 ] cluster (designated the SAM cluster) and a [Fe 4 S 4 ]-like cluster pair (designated the K cluster) that can be processed into an [Fe 8 S 9 ] precursor to the M cluster (designated the L cluster). Further, the K clusters in NifB Ma and NifB Mt can be converted to L clusters upon addition of SAM, which corresponds to their ability to heterologously donate L clusters to the biosynthetic machinery of A. vinelandii for further maturation into the M clusters. Perhaps even more excitingly, NifB Ma and NifB Mt can catalyze the removal of methyl group from SAM and the abstraction of hydrogen from this methyl group by 5′-deoxyadenosyl radical that initiates the radical-based incorporation of methyl-derived carbide into the M cluster. The successful identification of NifB Ma and NifB Mt as functional homologs of NifB not only enabled classification of a new subset of radical SAM methyltransferases that specialize in complex metallocluster assembly, but also provided a new tool for further characterization of the distinctive, NifB-catalyzed methyl transfer and conversion to an iron-bound carbide.nitrogenase | NifB | methanogens | radical SAM | homologs N itrogenase biosynthesis protein NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that plays an essential role in the biosynthesis of the M cluster, a [MoFe 8 S 9 C-homocitrate] cluster that serves as the cofactor of the molybdenum (Mo) nitrogenase from Azotobacter vinelandii (1-7). Carrying a signature CxxxCxxC motif at its N terminus that houses the SAM-binding [Fe 4 S 4 ] cluster (designated the SAM cluster), NifB also contains a number of additional ligands that could accommodate coordination of the entire complement of iron (Fe) atoms of the M cluster (Fig. S1). Moreover, it shares sequence homology with NifX, an accessory protein in M-cluster biosynthesis (8), toward its C terminus (Fig. S1). Characterization of the NifB protein from A. vinelandii had long been hampered by the instability of NifB in aqueous solutions until this protein was expressed as part of a NifEN-B fusion protein, wherein NifB was fused with and protected by NifEN, the biosynthetic apparatus immediately downstream of NifB along the M-cluster assembly pathway (9). Expression of the NifEN-B fusion protein in A. vinelendii was "modeled" after a naturally occurring NifEN-B fusion protein in Clostridium pasteurianum, which has the N terminus o...

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“…A new mechanism involving protein methylation and transitory cross-linking has recently been proposed to explain the mechanism of methylation by Cfr and RlmN (18)(19)(20). Also, an X-ray structure of RlmN with an Fe-S cluster, both with and without a SAM ligand, has been published (21). Cfr can be modeled on this structure, showing important differences between the enzymes (21).…”

mentioning

confidence: 99%

Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

Atkinson

Hansen

Tenson

et al. 2013

Antimicrob Agents Chemother

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Structural Basis for Methyl Transfer by a Radical SAM Enzyme

Cited by 158 publications

References 47 publications

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens

Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

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