Stable glycyl radical from pyruvate formate-lyase and ribonucleotide reductase (III)

Knappe, J; Af, Wagner

doi:10.1016/s0065-3233(01)58007-9

Cited by 34 publications

(22 citation statements)

References 87 publications

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“…A fourth ORF may provide the key to the biogenesis of CTQ, showing weak but significant homology to proteins that belong to a newly identified superfamily called the radical SAM proteins (22). These proteins have been shown to use an iron sulfur cluster to reductively cleave S-adenosylmethionine (SAM), producing a deoxyadenosyl radical center capable of initiating free radical conversions (23). These radical reactions include the formation of the glycyl radical found in pyruvate formate lyase (24) and the anaerobic ribonucleotide reductase (25).…”

Section: Figmentioning

confidence: 99%

How many ways to craft a cofactor?

Klinman

2001

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

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Section: Figmentioning

confidence: 99%

How many ways to craft a cofactor?

Klinman

2001

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

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“…Formate is formed in classical primary fermentations, such as the mixed‐acid fermentation by certain Enterobacteriaceae and by strict anaerobes in pyruvate cleavage by pyruvate formate lyase (Knappe and Wagner, ). Formate can be further converted by the same bacteria to H 2 + CO 2 by formate hydrogen lyase (Leonhartsberger et al ., ; Sinha et al ., ).…”

Section: Introductionmentioning

confidence: 99%

Hydrogen or formate: Alternative key players in methanogenic degradation

Schink

Montag

Keller

et al. 2017

Environ Microbiol Rep

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Hydrogen and formate are important electron carriers in methanogenic degradation in anoxic environments such as sediments, sewage sludge digestors and biogas reactors. Especially in the terminal steps of methanogenesis, they determine the energy budgets of secondary (syntrophically) fermenting bacteria and their methanogenic partners. The literature provides considerable data on hydrogen pool sizes in such habitats, but little data exist for formate concentrations due to technical difficulties in formate determination at low concentration. Recent evidence from biochemical and molecular biological studies indicates that several secondary fermenters can use both hydrogen and formate for electron release, and may do so even simultaneously. Numerous strictly anaerobic bacteria contain enzymes which equilibrate hydrogen and formate pools to energetically equal values, and recent measurements in sewage digestors and biogas reactors indicate that - beyond occasional fluctuations - the pool sizes of hydrogen and formate are indeed energetically nearly equivalent. Nonetheless, a thermophilic archaeon from a submarine hydrothermal vent, Thermococcus onnurineus, can obtain ATP from the conversion of formate to hydrogen plus bicarbonate at 80°C, indicating that at least in this extreme environment the pools of formate and hydrogen are likely to be sufficiently different to support such an unusual type of energy conservation.

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“…Several class II RS enzymes have been studied in vitro at varying levels of detail. The best characterized are the activating enzymes of the anaerobic ribonucleotide reductase and pyruvate formatelyase, both from E. coli (28,29,76,84).At the time of the early review, very little was known about class III enzymes. These enzymes cleave SAM irreversibly for each hydrogen atom abstracted by 5′-dA • in a given reaction sequence.…”

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In Vitro Characterization of AtsB, a Radical SAM Formylglycine-Generating Enzyme That Contains Three [4Fe-4S] Clusters

et al. 2008

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Sulfatases catalyze the cleavage of a variety of cellular sulfate esters via a novel mechanism that requires the action of a protein-derived formylglycine cofactor. Formation of the cofactor is catalyzed by an accessory protein and involves the two-electron oxidation of a specific cysteinyl or seryl residue on the relevant sulfatase. Although some sulfatases undergo maturation via mechanisms in which oxygen serves as an electron acceptor, AtsB, the maturase from Klebsiella pneumoniae, catalyzes the oxidation of Ser72 on AtsA, its cognate sulfatase, via an oxygen-independent mechanism. Moreover, it does not make use of pyridine or flavin nucleotide cofactors as direct electron acceptors. In fact, AtsB has been shown to be a member of the radical S-adenosylmethionine superfamily of proteins, suggesting that it catalyzes this oxidation via an intermediate 5′-deoxyadenosyl 5′-radical that is generated by a reductive cleavage of S-adenosyl-L-methionine. In contrast to AtsA, very little in vitro characterization of AtsB has been conducted. Herein we show that coexpression of the K. pneumoniae atsB gene with a plasmid that encodes genes that are known to be involved in ironsulfur cluster biosynthesis yields soluble protein that can be characterized in vitro. The as-isolated protein contained 8.7 ± 0.4 irons and 12.2 ± 2.6 sulfides per polypeptide, which existed almost entirely in the [4Fe-4S] 2+ configuration, as determined by Mössbauer spectroscopy, suggesting that it contained at least two of these clusters per polypeptide. Reconstitution of the as-isolated protein with additional iron and sulfide indicated the presence of 12.3 ± 0.2 irons and 9.9 ± 0.4 sulfides per polypeptide. Subsequent characterization of the reconstituted protein by Mössbauer spectroscopy indicated the presence of only [4Fe-4S] clusters, suggesting that reconstituted AtsB contains three per polypeptide. Consistent with this stoichiometry, an as-isolated AtsB triple variant containing Cys → Ala substitutions at each of the cysteines in its CX 3 CX 2 C radical SAM motif contained 7.3 ± 0.1 irons and 7.2 ± 0.2 sulfides per polypeptide while the reconstituted triple variant contained 7.7 ± 0.1 irons and 8.4 ± 0.4 sulfides per polypeptide, indicating that it was unable to incorporate an additional cluster. UV-visible and Mössbauer spectra of both samples indicated the presence of only clusters. AtsB was capable of catalyzing multiple turnovers and exhibited a V max /[E T ] of ~0.36 min −1 for an 18-amino acid peptide substrate using dithionite to supply the requisite electron and a value of ~0.039 min −1 for the same substrate using the physiologically relevant flavodoxin reducing system. Simultaneous quantification of formylglycine and 5′-deoxyadenosine as a function of time indicates an approximate 1:1 stoichiometry. Use of a peptide substrate in which the target serine is † This work was supported by NIH Grant GM-63847 (S.J.B.), the Dreyfus Foundation (Teacher Scholar Award to C.K.), and the Beckman Foundation (Young Investigator Award to...

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Stable glycyl radical from pyruvate formate-lyase and ribonucleotide reductase (III)

Cited by 34 publications

References 87 publications

How many ways to craft a cofactor?

How many ways to craft a cofactor?

Hydrogen or formate: Alternative key players in methanogenic degradation

In Vitro Characterization of AtsB, a Radical SAM Formylglycine-Generating Enzyme That Contains Three [4Fe-4S] Clusters

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