Structural Basis for Reductive Radical Formation and Electron Recycling in (<i>R</i>)-2-Hydroxyisocaproyl-CoA Dehydratase

Knauer, Stefan H.; Dobbek, Holger

doi:10.1021/ja1076537

Cited by 52 publications

(86 citation statements)

References 41 publications

(85 reference statements)

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“…Third, the cluster of TtuA is ligated by only three cysteines and thus, has a free coordination site on the fourth iron, which can be occupied by a hydrosulfide ligand. Precedents for exogenous sulfur species bound to an iron site of a [4Fe-4S] cluster have been reported: a comparable [4Fe-5S] cluster has been observed in HydG, an enzyme involved in maturation of hydrogenases (17), as well as 2-hydroxyisocaproyl-CoA dehydratase (18). Moreover, in the structure of the methylthiotransferase RimO, a polysulfide terminal ligand to a [4Fe-4S] cluster has been observed (19).…”

Section: Discussionmentioning

confidence: 95%

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster

Arragain

Bimaï

Legrand

et al. 2017

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

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Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA. Spectroscopic, enzymatic, and structural studies indicate that TtuA carries a catalytically essential [4Fe-4S] cluster and requires ATP for activity. A series of crystal structures shows that (i) the cluster is ligated by only three cysteines that are fully conserved, allowing the fourth unique iron to bind a small ligand, such as exogenous sulfide, and (ii) the ATP binding site, localized thanks to a proteinbound AMP molecule, a reaction product, is adjacent to the cluster. A mechanism for tRNA sulfuration is suggested, in which the unique iron of the catalytic cluster serves to bind exogenous sulfide, thus acting as a sulfur carrier.T he cellular translation machinery contains essential components such as tRNAs. To achieve their function, they feature a great variety of well-conserved posttranscriptional chemical modifications. Sulfur is present in several of these modified nucleosides: thiouridine and derivatives (s 4 U8, s 2 U34, and m 5 s 2 U54), 2-thioadenosine derivatives (ms 2 i 6 A37 and ms 2 t 6 A37), and 2-thiocytidine (s 2 C32). However, mechanisms of sulfur insertion into tRNAs are largely unknown, and the enzymes responsible for these reactions are incompletely characterized. Whereas redox conversion of a C-H to a C-S bond (synthesis of ms 2 i 6 A37 and ms 2 t 6 A37) depends on redox enzymes from the Radical-S-adenosyl-L-methionine iron-sulfur enzyme family, simple nonredox conversion of C = O to C = S group (synthesis of s 2 U34 and s 4 U8) is not expected to require such redox clusters. Intriguingly, we recently discovered that the ATPdependent formation of s 2 C32 in some tRNAs is catalyzed by an iron-sulfur enzyme, TtcA (1). However, the role of its cluster has not been defined. In the same superfamily, TtuA enzymes catalyze the C2-thiolation of uridine 54 in the T loop of thermophilic tRNAs (Fig. 1A), allowing stabilization of tRNAs at high temperature in thermophilic microorganisms. Sequences analysis shows that they share conserved cysteines and ATP binding motif (Fig. S1). Here, we report a detailed biochemical and structural characterization of TtuA that shows the presence of a [4Fe-4S] cluster essential for activity. The crystal structures of Pyrococcus horikoshii TtuA (PhTtuA) show that the cluster, chelated by only three cysteines, is adjacent to the ATP binding site. The presence of electron density near the fourth iron, nonbonded to the protein, indicates that the cluster can bind an exogenous substrate. We propose that thiolation occurs via sulfur binding to the cluster and transfer to the tRNA substrate. The fact that the catalytic [4Fe-4S] cluster serves as a sulfur carrier during a nonredox ...

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Section: Discussionmentioning

confidence: 95%

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster

Arragain

Bimaï

Legrand

et al. 2017

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

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“…T here are numerous examples where radicals play key roles in the active site of enzymes [1][2][3][4][5][6][7][8][9][10][11][12]. These radicals are normally located on a reactive amino acid side chain (cysteine [2], methionine, tyrosine, or tryptophan [10]) or on the peptide backbone, namely at the α-carbon positon of glycine [13].…”

Section: Introductionmentioning

confidence: 99%

“…These radicals are normally located on a reactive amino acid side chain (cysteine [2], methionine, tyrosine, or tryptophan [10]) or on the peptide backbone, namely at the α-carbon positon of glycine [13]. In some instances, it has also been shown that the radical migrates to different positions in the inactive form [4,5,9]. In those cases, the radical is relocated to the less reactive positions of the sulfur atom on a cysteine residue [4,5,9] or to the glycine α-carbon [6,11].…”

Section: Introductionmentioning

confidence: 99%

“…In some instances, it has also been shown that the radical migrates to different positions in the inactive form [4,5,9]. In those cases, the radical is relocated to the less reactive positions of the sulfur atom on a cysteine residue [4,5,9] or to the glycine α-carbon [6,11]. In most enzymes, such radical rearrangement is considered to be a protective mechanism.…”

Section: Introductionmentioning

confidence: 99%

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Structure and Reactivity of theN-Acetyl-Cysteine Radical Cation and Anion: Does Radical Migration Occur?

Osburn

Berden

O’Hair

et al. 2011

J. Am. Soc. Mass Spectrom.

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The structure and reactivity of the N-acetyl-cysteine radical cation and anion were studied using ion-molecule reactions, infrared multi-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations. The radical cation was generated by first nitrosylating the thiol of N-acetyl-cysteine followed by the homolytic cleavage of the S-NO bond in the gas phase. IRMPD spectroscopy coupled with DFT calculations revealed that for the radical cation the radical migrates from its initial position on the sulfur atom to the α-carbon position, which is 2.5 kJ mol -1 lower in energy. The radical migration was confirmed by time-resolved ion-molecule reactions. These results are in contrast with our previous study on cysteine methyl ester radical cation (Osburn et al., Chem. Eur. J. 2011, 17, 873-879) and the study by Sinha et al. for cysteine radical cation (Phys. Chem. Chem. Phys. 2010, 12, 9794-9800) where the radical was found to stay on the sulfur atom as formed. A similar approach allowed us to form a hydrogen-deficient radical anion of N-acetyl-cysteine, (M -2H)•-. IRMPD studies and ion-molecule reactions performed on the radical anion showed that the radical remains on the sulfur, which is the initial and more stable (by 63.6 kJ mol -1 ) position, and does not rearrange.

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“…The binding of two MgATP molecules to the reduced activator is supposed to induce a conformational change and drives formation of the complex with the dehydratase. ATP hydrolysis likely increases the reducing power of the reduced [4Fe-4S] cluster of the activator, enabling the one-electron transfer to the low-potential [4Fe-4S] cluster of the dehydratase (2,15). Unlike the 2-hydroxyacyl-CoA dehydratase system, the reduction of benzoyl-CoA is a two-step ETrequiring a stoichiometric consumption of ATP (3).…”

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confidence: 99%

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

Hennig

Jeoung

Goetzl

et al. 2012

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

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Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E 0 0 < −500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/ sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the [Ni-Ni-Fe 4 S 4 ] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the lowpotential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like [2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins.

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Structural Basis for Reductive Radical Formation and Electron Recycling in (R)-2-Hydroxyisocaproyl-CoA Dehydratase

Cited by 52 publications

References 41 publications

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster

Structure and Reactivity of theN-Acetyl-Cysteine Radical Cation and Anion: Does Radical Migration Occur?

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

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