On the Role of Additional [4Fe-4S] Clusters with a Free Coordination Site in Radical-SAM Enzymes

Mulliez, Etienne; Duarte, Victor; Arragain, Simon; Fontecave, Marc; Atta, Mohamed

doi:10.3389/fchem.2017.00017

Cited by 33 publications

(27 citation statements)

References 72 publications

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“…2c). The substrate binding manner of [4Fe-4S] cluster in TtuA is similar to that of aconitase 28 and radical-S-adenosyl-L-methionine enzymes 29 , which use the unique Fe site to place a water molecule necessary for the reaction at an appropriate position. Along the axis of the octahedron, a water molecule was detected~3.7 Å from the unique Fe site (Fig.…”

Section: Resultsmentioning

confidence: 89%

The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus

et al. 2020

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TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s 2 T) at position 54 of transfer RNA (tRNA), which is responsible for adaptation to high temperature environments in Thermus thermophilus. The enzymatic activity of TtuA requires an iron-sulfur (Fe-S) cluster, by which a sulfur atom supplied by TtuB is transferred to the tRNA substrate. Here, we demonstrate that the Fe-S cluster directly receives sulfur from TtuB through its inherent coordination ability. TtuB forms a [4Fe-4S]-TtuB intermediate, but that sulfur is not immediately released from TtuB. Further desulfurization assays and mutation studies demonstrated that the release of sulfur from the thiocarboxylated C-terminus of TtuB is dependent on adenylation of the substrate tRNA, and the essential residue for TtuB desulfurization was identified. Based on these findings, the molecular mechanism of sulfur transfer from TtuB to Fe-S cluster is proposed.

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Section: Resultsmentioning

confidence: 89%

The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus

et al. 2020

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“…However, although NirJ possesses all four cysteines for the ligation of the Aux II cluster, the presence of which was suggested in this study, NirJ lacks the cysteines required for binding the Aux I cluster of SPASM enzymes. In some cases they serve as a source of sulfur, in others they are required for substrate binding or for electron transfer [26,27]. At present, the role of the auxiliary cluster in NirJ is not known.…”

Section: Resultsmentioning

confidence: 99%

“…The roles of additional iron-sulfur clusters in Radical SAM enzymes (SPASM and non-SPASM) are quite diverse. In some cases they serve as a source of sulfur, in others they are required for substrate binding or for electron transfer [26,27]. For anSMEs, which catalyze a dehydrogenation reaction, an electron transfer function was proposed for the auxiliary clusters [28].…”

Section: Nirj Contains Two Iron-sulfur Clustersmentioning

confidence: 99%

The Radical SAM enzyme NirJ catalyzes the removal of two propionate side chains during heme d₁ biosynthesis

et al. 2017

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Heme d is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd nitrite reductase of many denitrifying bacteria. In the course of heme d biosynthesis, the two propionate side chains on pyrrole rings A and B of the intermediate 12,18-didecarboxysiroheme are removed from the tetrapyrrole macrocycle. In the final heme d molecule, the propionate groups are replaced by two keto functions. Although it was speculated that the Radical S-adenosyl-l-methionine (SAM) enzyme NirJ might be responsible for the removal of the propionate groups and introduction of the keto functions, this has not been shown experimentally, so far. Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron-sulfur clusters. While the N-terminal [4Fe-4S] cluster is essential for the initial SAM cleavage reaction, it is not required for substrate binding. NirJ tightly binds its substrate 12,18-didecarboxysiroheme and, thus, can be purified in complex with the substrate. By using the purified NirJ/substrate complex in an in vitro enzyme activity assay, we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. However, under the reaction conditions employed, no keto group formation is observed indicating that an additional cofactor or enzyme is needed for this reaction.

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“…These metallic active sites are flexible in terms of the number and nature of the ligands and the coordination geometries. A prime example of this versatility is given by the large family of proteins hosting iron−sulfur clusters of a variety of forms: while the most common ligand of the iron ions in iron−sulfur clusters are cysteins, other ligands are found like histidines, aspartate, arginine, threonine, water molecules, or even no ligand, for instance in the case of radical SAM enzymes, which harbor a vacant coordination position where the substrate binds …”

Section: Introductionmentioning

confidence: 99%

Redox (In)activations of Metalloenzymes: A Protein Film Voltammetry Approach

Barrio

Fourmond

2019

ChemElectroChem

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Redox metalloenzymes are omnipresent in living organisms where they catalyze key cellular reactions with great efficiency. These enzymes can often be reversibly placed into inactive states following changes in redox conditions. This is a hindrance for their use in biotechnological devices, and also a complication for their study via a structure/function approach, because structural data alone usually is not enough to discriminate between active and inactive states. However, these inactive states can also inform on the chemistry of the enzyme's active sites and on their catalytic cycles. A technique that has proved particularly valuable in the last decades for studying these processes is protein film voltammetry (PFV), in which an enzyme is immobilized on an electrode in a configuration where direct electron transfer is possible. In this article, we review the studies of redox (in)activation processes using PFV, present the theory for a number of cases (reversible inactivations, irreversible activations), and give guidelines to obtain and interpret suitable kinetic data.[a] Dr. M. del Barrio, Dr. V. Fourmond

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On the Role of Additional [4Fe-4S] Clusters with a Free Coordination Site in Radical-SAM Enzymes

Cited by 33 publications

References 72 publications

The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus

The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus

The Radical SAM enzyme NirJ catalyzes the removal of two propionate side chains during heme d₁ biosynthesis

Redox (In)activations of Metalloenzymes: A Protein Film Voltammetry Approach

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On the Role of Additional [4Fe-4S] Clusters with a Free Coordination Site in Radical-SAM Enzymes

Cited by 33 publications

References 72 publications

The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus

The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus

The Radical SAM enzyme NirJ catalyzes the removal of two propionate side chains during heme d1 biosynthesis

Redox (In)activations of Metalloenzymes: A Protein Film Voltammetry Approach

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The Radical SAM enzyme NirJ catalyzes the removal of two propionate side chains during heme d₁ biosynthesis