The B12-independent glycerol dehydratase activating enzyme from Clostridium butyricum cleaves SAM to produce 5′-deoxyadenosine and not 5′-deoxy-5′-(methylthio)adenosine

Walls, William G.; Moody, J. D.; McDaniel, Elizabeth C.; Villanueva, María; Shepard, Eric M.; Broderick, William E.; Broderick, Joan B.

doi:10.1016/j.jinorgbio.2021.111662

Cited by 16 publications

(16 citation statements)

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“…Natural abundant and isotopically labeled SAM were synthesized as previously described. , The purification of SAM was carried out following a previously published protocol …”

Section: Methodsmentioning

confidence: 99%

“…Radical S -adenosyl- l -methionine (SAM) enzymes are found in all domains of life and catalyze an astounding array of reactions, some of which have potential applications in the biosynthesis of pharmaceutically or technologically useful compounds, such as antibiotics or biofuels. − Radical SAM (RS) enzymes have a partial or full TIM barrel fold structure and bind a site-differentiated [4Fe–4S] cluster with three cysteines from a conserved CX 3 CX 2 C motif . The unique iron of the [4Fe–4S] cluster is coordinated by the amino and carboxylate moieties of SAM to form a coordination complex central to the catalytic mechanism. − Catalysis by RS enzymes is initiated via electron transfer from the reduced [4Fe–4S] 1+ cluster to the sulfonium of SAM, , resulting in reductive cleavage of the S–C5′ bond of SAM; − this reductive cleavage leads to the formation of an organometallic intermediate Ω in which the 5′-C of the 5′-deoxyadenosyl fragment is directly bound to the unique iron of the [4Fe–4S] cluster. , While initially surprising, Ω has been observed in a wide range of RS enzymes − and is proposed to be a key intermediate across the RS superfamily . Synthetic models of Ω developed by Suess and coworkers have provided further insight into this species. − In the reaction of the RS enzyme pyruvate formate-lyase activating enzyme (PFL-AE) with its substrate pyruvate formate-lyase (PFL), Ω is shown to convert cleanly to the product glycyl radical upon thermal annealing, demonstrating the catalytic competence of Ω .…”

Section: Introductionmentioning

confidence: 99%

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Mechanism of Radical S-Adenosyl-l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5′-Deoxyadenosyl Radical

et al. 2022

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Radical S-adenosyl-l-methionine (SAM) enzymes employ a [4Fe–4S] cluster and SAM to initiate diverse radical reactions via either H-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR) spectroscopy to provide snapshots of the reaction pathway in an adenosylation reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme on a peptide substrate containing a dehydroalanine residue in place of the target glycine. The reaction proceeds via the initial formation of the organometallic intermediate Ω, as evidenced by the characteristic EPR signal with g ∥ = 2.035 and g ⊥ = 2.004 observed when the reaction is freeze-quenched at 500 ms. Thermal annealing of frozen Ω converts it into a second paramagnetic species centered at g iso = 2.004; this second species was generated directly using freeze-quench at intermediate times (∼8 s) and unequivocally identified via isotopic labeling and EPR spectroscopy as the tertiary peptide radical resulting from adenosylation of the peptide substrate. An additional paramagnetic species observed in samples quenched at intermediate times was revealed through thermal annealing while frozen and spectral subtraction as the SAM-derived 5′-deoxyadenosyl radical (5′-dAdo•). The time course of the 5′-dAdo• and tertiary peptide radical EPR signals reveals that the former generates the latter. These results thus support a mechanism in which Ω liberates 5′-dAdo• by Fe–C5′ bond homolysis, and the 5′-dAdo• attacks the dehydroalanine residue of the peptide substrate to form the adenosylated peptide radical species. The results thus provide a picture of a catalytically competent 5′-dAdo• intermediate trapped just prior to reaction with the substrate.

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“…Natural abundant and isotopically labeled SAM were synthesized as previously described. , The purification of SAM was carried out following a previously published protocol …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mechanism of Radical S-Adenosyl-l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5′-Deoxyadenosyl Radical

et al. 2022

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“…Interestingly, the special SAM cleavage activity of Dph2 resulted from the distinct geometry of SAM binding with 4Fe-4S, which was conjugated by three cysteines from different domains of the protein instead of the CxxxCxxC motif. 25 The Broderick group 27 and our group 28 recently demonstrated that glycerol dehydrogenase-activating enzyme GD-AE, the only previously assumed noncanonical radical SAM enzyme with the CxxxCxxC motif, is a canonical radical SAM enzyme that generates the 5′-dA radical. Moreover, many enzymes, such as TWY2 29 , Tsr3 30 and AzeJ 31 , cleave the C γ,Met -S bond of SAM by a nucleophilic mechanism.…”

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confidence: 99%

Arsinothricin Biosynthesis Involving a Noncanonical Radical SAM Enzyme for C-As Bond Formation

Yao,

He,

Dong

2024

Preprint

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Arsinothricin is a potent antibiotic secreted by soil bacteria. The biosynthesis of Arsinothricin was proposed to involve two steps. The first step is C–As bond formation between trivalent As and the 3–amino–3–carboxypropyl (ACP) group of S–adenosyl–L–methionine (SAM), which is catalyzed by the protein ArsL. However, the reaction has not been verified in vitro, and ArsL has not been characterized in detail. Interestingly, ArsL contains a CxxxCxxC motif and thus belongs to the radical SAM enzyme superfamily, the members of which cleave SAM and generate a 5′–deoxyadenosyl radical. Here, we found that ArsL cleaves the Cγ,Met–S bond of SAM and generates an ACP radical that resembles Dph2, a noncanonical radical SAM enzyme involved in diphthamid biosynthesis. As Dph2 does not contain the CxxxCxxC motif, ArsL is a unique noncanonical radical SAM enzyme that contains this motif but generates an ACP radical. Together with the methyltransferase ArsM, we successfully reconstituted arsinothricin biosynthesis in vitro. ArsL has a conserved RCCLKC motif in the C–terminal sequence and belongs to the RCCLKC–tail radical SAM protein subfamily. By truncation, we showed that this motif binds to the substrate arsenite and is highly important for its activity. Our results suggested that ArsL is a noncanonical radical SAM enzyme with a canonical radical SAM enzyme motif, implying that more noncanonical radical SAM chemistry may exist within the radical SAM enzyme superfamily

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“…During the review of this work, the Broderick group also reported that GD-AE actually cleaves SAM to produce 5′-dA. 32…”

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confidence: 95%

Mechanistic investigation of B12-independent glycerol dehydratase and its activating enzyme GD-AE

Yao

et al. 2022

Chem. Commun.

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The B12-independent glycerol dehydratase activating enzyme from Clostridium butyricum cleaves SAM to produce 5′-deoxyadenosine and not 5′-deoxy-5′-(methylthio)adenosine

Cited by 16 publications

References 55 publications

Mechanism of Radical S-Adenosyl-l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5′-Deoxyadenosyl Radical

Mechanism of Radical S-Adenosyl-l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5′-Deoxyadenosyl Radical

Arsinothricin Biosynthesis Involving a Noncanonical Radical SAM Enzyme for C-As Bond Formation

Mechanistic investigation of B12-independent glycerol dehydratase and its activating enzyme GD-AE

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