Structure-based insights into the mechanism of [4Fe-4S]-dependent sulfur insertase LarE.

Several essential cellular metabolites, such as enzyme cofactors, contain sulfur atoms and their biosynthesis requires specific thiolation enzymes. LarE is an ATP-dependent sulfur insertase, which catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two types of LarE enzymes are known, one that uses a catalytic cysteine as a sacrificial sulfur donor, and the other one that uses a [4Fe-4S] cluster as a cofactor. Only the crystal structure of LarE from Lactobacillus plantarum (LpLarE) from the first class has been solved. We report here the crystal structure of LarE from Methanococcus maripaludis (MmLarE), belonging to the second class, in the cluster-free (apo-) and cluster-bound (holo-) forms. The structure of holo-MmLarE shows that the [4Fe-4S] cluster is chelated by three cysteines only, leaving an open coordination site on one Fe atom. Moreover, the fourth nonprotein-bonded iron atom was able to bind an anionic ligand such as a phosphate group or a chloride ion. Together with the spectroscopic analysis of holo-MmLarE and the previously reported biochemical investigations of holo-LarE from Thermotoga maritima, these crystal structures support the hypothesis of a reaction mechanism, in which the [4Fe-4S] cluster binds a hydrogenosulfide ligand in place of the chloride anion, thus generating a [4Fe-5S] intermediate, and transfers it to the substrate, as in the case of [4Fe-4S]-dependent tRNA thiolation enzymes.

Silver and Copper Nitride Cooperate for CO Electroreduction to Propanol.

The need of carbon sources for the chemical industry, alternative to fossil sources, has pointed to CO2 as a possible feedstock. While CO2 electroreduction (CO2 R) allows production of interesting organic compounds, it suffers from large carbon losses, mainly due to carbonate formation. This is why, quite recently, tandem CO2 R, a two-step process, with first CO2 R to CO using a solid oxide electrolysis cell followed by CO electroreduction (COR), has been considered, since no carbon is lost as carbonate in either step. Here we report a novel copper-based catalyst, silver-doped copper nitride, with record selectivity for formation of propanol (Faradaic efficiency: 45 %), an industrially relevant compound, from CO electroreduction in gas-fed flow cells. Selective propanol formation occurs at metallic copper atoms derived from copper nitride and is promoted by silver doping as shown experimentally and computationally. In addition, the selectivity for C2+ liquid products (Faradaic efficiency: 80 %) is among the highest reported so far. These findings open new perspectives regarding the design of catalysts for production of C3 compounds from CO2 .

The thiolation of uridine 34 in tRNA, which controls protein translation, depends on a [4Fe-4S] cluster in the archaeum Methanococcus maripaludis.

Thiolation of uridine 34 in the anticodon loop of several tRNAs is conserved in the three domains of life and guarantees fidelity of protein translation. U34-tRNA thiolation is catalyzed by a complex of two proteins in the eukaryotic cytosol (named Ctu1/Ctu2 in humans), but by a single NcsA enzyme in archaea. We report here spectroscopic and biochemical experiments showing that NcsA from Methanococcus maripaludis (MmNcsA) is a dimer that binds a [4Fe-4S] cluster, which is required for catalysis. Moreover, the crystal structure of MmNcsA at 2.8 Å resolution shows that the [4Fe-4S] cluster is coordinated by three conserved cysteines only, in each monomer. Extra electron density on the fourth nonprotein-bonded iron most likely locates the binding site for a hydrogenosulfide ligand, in agreement with the [4Fe-4S] cluster being used to bind and activate the sulfur atom of the sulfur donor. Comparison of the crystal structure of MmNcsA with the AlphaFold model of the human Ctu1/Ctu2 complex shows a very close superposition of the catalytic site residues, including the cysteines that coordinate the [4Fe-4S] cluster in MmNcsA. We thus propose that the same mechanism for U34-tRNA thiolation, mediated by a [4Fe-4S]-dependent enzyme, operates in archaea and eukaryotes.

Identification of 2-Methylthio-methylenethio-N6 -(cis-4-hydroxyisopentenyl)adenosine (msms2 io6 A37 ) as a Novel Modification at Adenosine 37 of tRNAs from Salmonella typhimurium.

Post-transcriptional modifications of tRNA nucleotide are important determinants in folding, structure and function. We have successfully identified and characterized a new modified base named 2-methylthio-methylenethio-N6 -(cis-4-hydroxyisopentenyl)adenosine, which is present at position 37 in some tRNAs. We also showed that this new modified adenosine is derived from the known 2-methylthio-methylenethio-N6 -(isopentenyl)adenosine nucleoside by a catalytic cycle of the tRNA-diiron monooxygenase, MiaE, present in Salmonella typhimurium.

A subclass of archaeal U8-tRNA sulfurases requires a [4Fe-4S] cluster for catalysis.

Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC + C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe-4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe-4S] cluster is coordinated by the three cysteines of the CXXC + C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.

CO2 Electroreduction in Water with a Heterogenized C-Substituted Nickel Cyclam Catalyst.

Molecular catalysis for selective CO2 electroreduction into CO can be achieved with a variety of metal complexes. Their immobilization on cathodes is required for their practical implementation in electrolytic cells and can benefit from the advantages of a solid material such as easy separation of products and catalysts, efficient electron transfer to the catalyst, and high stability. However, this approach remains insufficiently explored up to now. Here, using an appropriate and original modification of the cyclam ligand, we report a novel [Ni(cyclam)]2+ complex which can be immobilized on carbon nanotubes. This material, once deposited on a gas diffusion layer, provides a novel electrode which is remarkably selective for CO2 electroreduction to CO, not only in organic solvents but also, more remarkably, in water, with faradic efficiencies for CO larger than 90% and current densities of 5-10 mA cm-2 during controlled potential electrolysis in H-cells.

Selective Ethylene Production from CO2 and CO Reduction via Engineering Membrane Electrode Assembly with Porous Dendritic Copper Oxide.

Gas-fed zero-gap electrolyzers have recently emerged as attractive systems for limiting ohmic losses and costs associated with electrolytes and for optimizing energy efficiencies. Here, we report that using a dendritic Cu oxide (D-CuO) material deposited on a gas diffusion layer as the cathode of a gas-fed zero-gap membrane electrode assembly (MEA) system results in a very selective conversion of CO to ethylene. More specifically, CO reduction yielded ethylene with an FE up to 68% at 100-125 mA·cm-2 with H2 as the only other gaseous product and the electrolysis could be carried out for several hours with good stability. Ethylene was also the major product during CO2 electrolysis (FE = 41%) at 125-150 mA·cm-2, reflecting the high selectivity of D-CuO for ethylene production. Such systems are relevant for tandem CO2 electroreduction processes, allowing energy efficiencies above 30%.

An enzymatic activation of formaldehyde for nucleotide methylation.

Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

Iron-sulfur biology invades tRNA modification: the case of U34 sulfuration.

Sulfuration of uridine 34 in the anticodon of tRNAs is conserved in the three domains of life, guaranteeing fidelity of protein translation. In eubacteria, it is catalyzed by MnmA-type enzymes, which were previously concluded not to depend on an iron-sulfur [Fe-S] cluster. However, we report here spectroscopic and iron/sulfur analysis, as well as in vitro catalytic assays and site-directed mutagenesis studies unambiguously showing that MnmA from Escherichia coli can bind a [4Fe-4S] cluster, which is essential for sulfuration of U34-tRNA. We propose that the cluster serves to bind and activate hydrosulfide for nucleophilic attack on the adenylated nucleoside. Intriguingly, we found that E. coli cells retain s2U34 biosynthesis in the ΔiscUA ΔsufABCDSE strain, lacking functional ISC and SUF [Fe-S] cluster assembly machineries, thus suggesting an original and yet undescribed way of maturation of MnmA. Moreover, we report genetic analysis showing the importance of MnmA for sustaining oxidative stress.

Structural Evidence for a [4Fe-5S] Intermediate in the Non-Redox Desulfuration of Thiouracil.

We recently discovered a [Fe-S]-containing protein with in vivo thiouracil desulfidase activity, dubbed TudS. The crystal structure of TudS refined at 1.5 Šresolution is reported; it harbors a [4Fe-4S] cluster bound by three cysteines only. Incubation of TudS crystals with 4-thiouracil trapped the cluster with a hydrosulfide ligand bound to the fourth non-protein-bonded iron, as established by the sulfur anomalous signal. This indicates that a [4Fe-5S] state of the cluster is a catalytic intermediate in the desulfuration reaction. Structural data and site-directed mutagenesis indicate that a water molecule is located next to the hydrosulfide ligand and to two catalytically important residues, Ser101 and Glu45. This information, together with modeling studies allow us to propose a mechanism for the unprecedented non-redox enzymatic desulfuration of thiouracil, in which a [4Fe-4S] cluster binds and activates the sulfur atom of the substrate.

Functionalization of Carbon Nanotubes with Nickel Cyclam for the Electrochemical Reduction of CO2.

The exploitation of molecular catalysts for CO2 electrolysis requires their immobilization on the cathode of the electrolyzer. As an illustration of this approach, a Ni-cyclam complex with a cyclam derivative functionalized with a pyrene moiety is synthesized, found to be a selective catalyst for CO2 electroreduction to CO, and immobilized on a carbon nanotube-coated gas diffusion electrode by using a noncovalent binding strategy. The as-prepared electrode is efficient, selective, and robust for electrocatalytic reduction of CO2 to CO. Very high turnover numbers (ca. 61460) and turnover frequencies (ca. 4.27 s-1 ) are enabled by the novel electrode material in organic solvent-water mixtures saturated with CO2 . This material provides an interesting platform for further improvement.

Structural, biochemical and functional analyses of tRNA-monooxygenase enzyme MiaE from Pseudomonas putida provide insights into tRNA/MiaE interaction.

MiaE (2-methylthio-N6-isopentenyl-adenosine37-tRNA monooxygenase) is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide 2-methylthio-N6-isopentenyl-adenosine (ms2i6A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N6-4-hydroxyisopentenyl-adenosine (ms2io6A37). Here, we report the in vivo activity, biochemical, spectroscopic characterization and X-ray crystal structure of MiaE from Pseudomonas putida. The investigation demonstrates that the putative pp-2188 gene encodes a MiaE enzyme. The structure shows that Pp-MiaE consists of a catalytic diiron(III) domain with a four alpha-helix bundle fold. A docking model of Pp-MiaE in complex with tRNA, combined with site directed mutagenesis and in vivo activity shed light on the importance of an additional linker region for substrate tRNA recognition. Finally, krypton-pressurized Pp-MiaE experiments, revealed the presence of defined O2 site along a conserved hydrophobic tunnel leading to the diiron active center.

The O2-independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa.

Many proteobacteria, such as Escherichia coli, contain two main types of quinones: benzoquinones, represented by ubiquinone (UQ) and naphthoquinones, such as menaquinone (MK), and dimethyl-menaquinone (DMK). MK and DMK function predominantly in anaerobic respiratory chains, whereas UQ is the major electron carrier in the reduction of dioxygen. However, this division of labor is probably not very strict. Indeed, a pathway that produces UQ under anaerobic conditions in an UbiU-, UbiV-, and UbiT-dependent manner has been discovered recently in E. coli Its physiological relevance is not yet understood, because MK and DMK are also present in E. coli Here, we established that UQ9 is the major quinone of Pseudomonas aeruginosa and is required for growth under anaerobic respiration (i.e. denitrification). We demonstrate that the ORFs PA3911, PA3912, and PA3913, which are homologs of the E. coli ubiT, ubiV, and ubiU genes, respectively, are essential for UQ9 biosynthesis and, thus, for denitrification in P. aeruginosa These three genes here are called ubiTPa , ubiVPa , and ubiUPa We show that UbiVPa accommodates an iron-sulfur [4Fe-4S] cluster. Moreover, we report that UbiUPa and UbiTPa can bind UQ and that the isoprenoid tail of UQ is the structural determinant required for recognition by these two Ubi proteins. Since the denitrification metabolism of P. aeruginosa is believed to be important for the pathogenicity of this bacterium in individuals with cystic fibrosis, our results highlight that the O2-independent UQ biosynthetic pathway may represent a target for antibiotics development to manage P. aeruginosa infections.

Structural and Functional Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase from Escherichia coli.

The hydroxylation of phenols into polyphenols, which are valuable chemicals and pharmaceutical products, is a challenging reaction. The search for green synthetic processes has led to considering microorganisms and pure hydroxylases as catalysts for phenol hydroxylation. Herein, we report the structural and functional characterization of the flavin adenine dinucleotide (FAD)-dependent 4-hydroxyphenylacetate 3-monooxygenase from Escherichia coli, named HpaB. It is shown that this enzyme enjoys a relatively broad substrate specificity, which allows the conversion of a number of non-natural phenolic compounds, such as tyrosol, hydroxymandelic acid, coumaric acid, hydroxybenzoic acid and its methyl ester, and phenol, into the corresponding catechols. The reaction can be performed by using a simple chemical assay based on formate as the electron donor and the organometallic complex [Rh(bpy)Cp*(H2 O)]2+ (Cp*: 1,2,3,4,5-pentamethylcyclopentadiene, bpy: 2,2'-bipyridyl) as the catalyst for FAD reduction. The availability of a crystal structure of HpaB in complex with FAD at 1.8 Šresolution opens up the possibility of the rational tuning of the substrate specificity and activity of this interesting class of phenol hydroxylases.

Carbon-Nanotube-Supported Copper Polyphthalocyanine for Efficient and Selective Electrocatalytic CO2 Reduction to CO.

Electroreduction of CO2 to CO is one of the simplest ways to valorise CO2 as a source of carbon. Herein, a cheap, robust, Cu-based hybrid catalyst consisting of a polymer of Cu phthalocyanine coated on carbon nanotubes, which proved to be selective for CO production (80 % faradaic yield) at relatively low overpotentials, was developed. Polymerisation of Cu phthalocyanine was shown to have a drastic effect on the selectivity of the reaction because molecular Cu phthalocyanine was instead selective for proton reduction under the same conditions. Although the material only showed isolated Cu sites in phthalocyanine-like CuN4 coordination, in situ and operando X-ray absorption spectroscopy showed that, under operating conditions, the Cu atoms were fully converted to Cu nanoparticles, which were likely the catalytically active species. Interestingly, this restructuring of the metal sites was reversible.

Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin.

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.

Ubiquinone Biosynthesis over the Entire O2 Range: Characterization of a Conserved O2-Independent Pathway.

Most bacteria can generate ATP by respiratory metabolism, in which electrons are shuttled from reduced substrates to terminal electron acceptors, via quinone molecules like ubiquinone. Dioxygen (O2) is the terminal electron acceptor of aerobic respiration and serves as a co-substrate in the biosynthesis of ubiquinone. Here, we characterize a novel, O2-independent pathway for the biosynthesis of ubiquinone. This pathway relies on three proteins, UbiT (YhbT), UbiU (YhbU), and UbiV (YhbV). UbiT contains an SCP2 lipid-binding domain and is likely an accessory factor of the biosynthetic pathway, while UbiU and UbiV (UbiU-UbiV) are involved in hydroxylation reactions and represent a novel class of O2-independent hydroxylases. We demonstrate that UbiU-UbiV form a heterodimer, wherein each protein binds a 4Fe-4S cluster via conserved cysteines that are essential for activity. The UbiT, -U, and -V proteins are found in alpha-, beta-, and gammaproteobacterial clades, including several human pathogens, supporting the widespread distribution of a previously unrecognized capacity to synthesize ubiquinone in the absence of O2 Together, the O2-dependent and O2-independent ubiquinone biosynthesis pathways contribute to optimizing bacterial metabolism over the entire O2 range.IMPORTANCE In order to colonize environments with large O2 gradients or fluctuating O2 levels, bacteria have developed metabolic responses that remain incompletely understood. Such adaptations have been recently linked to antibiotic resistance, virulence, and the capacity to develop in complex ecosystems like the microbiota. Here, we identify a novel pathway for the biosynthesis of ubiquinone, a molecule with a key role in cellular bioenergetics. We link three uncharacterized genes of Escherichia coli to this pathway and show that the pathway functions independently from O2 In contrast, the long-described pathway for ubiquinone biosynthesis requires O2 as a substrate. In fact, we find that many proteobacteria are equipped with the O2-dependent and O2-independent pathways, supporting that they are able to synthesize ubiquinone over the entire O2 range. Overall, we propose that the novel O2-independent pathway is part of the metabolic plasticity developed by proteobacteria to face various environmental O2 levels.

A Soluble Metabolon Synthesizes the Isoprenoid Lipid Ubiquinone.

Ubiquinone (UQ) is a polyprenylated lipid that is conserved from bacteria to humans and is crucial to cellular respiration. How the cell orchestrates the efficient synthesis of UQ, which involves the modification of extremely hydrophobic substrates by multiple sequential enzymes, remains an unresolved issue. Here, we demonstrate that seven Ubi proteins form the Ubi complex, a stable metabolon that catalyzes the last six reactions of the UQ biosynthetic pathway in Escherichia coli. The SCP2 domain of UbiJ forms an extended hydrophobic cavity that binds UQ intermediates inside the 1-MDa Ubi complex. We purify the Ubi complex from cytoplasmic extracts and demonstrate that UQ biosynthesis occurs in this fraction, challenging the current thinking of a membrane-associated biosynthetic process. Collectively, our results document a rare case of stable metabolon and highlight how the supramolecular organization of soluble enzymes allows the modification of hydrophobic substrates in a hydrophilic environment.

Power of protein/tRNA functional assembly against aberrant aggregation.

Understanding the mechanisms of protein oligomerization and aggregation is a major concern for biotechnology and medical purposes. However, significant challenges remain in determining the mechanism of formation of these superstructures and the environmental factors that can precisely modulate them. Notably the role that a functional ligand plays in the process of protein aggregation is largely unexplored. We herein address these issues with an original flavin-dependent RNA methyltransferase (TrmFO) used as a protein model since this protein employs a complex set of cofactors and ligands for catalysis. Here, we show that TrmFO carries an unstable protein structure that can partially mis-unfold leading to either formation of irregular and nonfunctional soluble oligomers endowed with hyper-thermal stability or large amorphous aggregates in the presence of salts. Mutagenesis confirmed that this peculiarity is an intrinsic property of a polypeptide and it is independent of the flavin coenzyme. Structural characterization and kinetic studies identified several regions of the protein that enjoy conformational changes and more particularly pinpointed the N-terminal subdomain as being a key element in the mechanisms of oligomerization and aggregation. Only stabilization of this region via tRNA suppresses these aberrant protein states. Although protein chaperones emerged as major actors against aggregation, our study emphasizes that other powerful mechanisms exist such as the stabilizing effect of functional assemblies that provide an additional layer of protection against the instability of the proteome.