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.

Integrative Approach to Probe Alternative Redox Mechanisms in RNA Modifications.

RNA modifications found in most RNAs, particularly in tRNAs and rRNAs, reveal an abundance of chemical alterations of nucleotides. Over 150 distinct RNA modifications are known, emphasizing a remarkable diversity of chemical moieties in RNA molecules. These modifications play pivotal roles in RNA maturation, structural integrity, and the fidelity and efficiency of translation processes.

Oxygen-induced chromophore degradation in the photoswitchable red fluorescent protein rsCherry.

Reversibly switchable monomeric Cherry (rsCherry) is a photoswitchable variant of the red fluorescent protein mCherry. We report that this protein gradually and irreversibly loses its red fluorescence in the dark over a period of months at 4 °C and a few days at 37 °C. We also find that its ancestor, mCherry, undergoes a similar fluorescence loss but at a slower rate.

Evolutionary Diversity of Dus2 Enzymes Reveals Novel Structural and Functional Features among Members of the RNA Dihydrouridine Synthases Family.

Dihydrouridine (D) is an abundant modified base found in the tRNAs of most living organisms and was recently detected in eukaryotic mRNAs. This base confers significant conformational plasticity to RNA molecules. The dihydrouridine biosynthetic reaction is catalyzed by a large family of flavoenzymes, the dihydrouridine synthases (Dus).

Dihydrouridine in the Transcriptome: New Life for This Ancient RNA Chemical Modification.

Until recently, post-transcriptional modifications of RNA were largely restricted to noncoding RNA species. However, this belief seems to have quickly dissipated with the growing number of new modifications found in mRNA that were originally thought to be primarily tRNA-specific, such as dihydrouridine. Recently, transcriptomic profiling, metabolic labeling, and proteomics have identified unexpected dihydrouridylation of mRNAs, greatly expanding the catalog of novel mRNA modifications.

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.

Dihydrouridine synthesis in tRNAs is under reductive evolution in Mollicutes.

Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in .

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 Å resolution is reported; it harbors a [4Fe-4S] cluster bound by three cysteines only.

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.

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.

Conformational Stability Adaptation of a Double-Stranded RNA-Binding Domain to Transfer RNA Ligand.

The double-stranded RNA-binding domain (dsRBD) is a broadly distributed domain among RNA-maturing enzymes. Although this domain recognizes dsRNA's structures via a conserved canonical structure adopting an α-βββ-α topology, several dsRBDs can accommodate discrete structural extensions expanding further their functional repertoire. How these structural elements engage cooperative communications with the canonical structure and how they contribute to the dsRBD's overall folding are poorly understood.

Shigella IpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion.

The Shigella type III effector IpaA contains three binding sites for the focal adhesion protein vinculin (VBSs), which are involved in bacterial invasion of host cells. Here, we report that IpaA VBS3 unexpectedly binds to talin. The 2.

Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2.

Double stranded RNA-binding domain (dsRBD) is a ubiquitous domain specialized in the recognition of double-stranded RNAs (dsRNAs). Present in many proteins and enzymes involved in various functional roles of RNA metabolism, including RNA splicing, editing, and transport, dsRBD generally binds to RNAs that lack complex structures. However, this belief has recently been challenged by the discovery of a dsRBD serving as a major tRNA binding module for human dihydrouridine synthase 2 (hDus2), a flavoenzyme that catalyzes synthesis of dihydrouridine within the complex elbow structure of tRNA.

Electrostatic Potential in the tRNA Binding Evolution of Dihydrouridine Synthases.

Dihydrouridine (D) is an abundant modified base of tRNA found in the majority of living organisms. This base is synthesized via an NADPH-dependent reduction of specific uridines by the dihydrouridine synthases (Dus), a large family of flavoenzymes comprising eight subfamilies. Almost all of these enzymes function with only two conserved domains, an N-terminal catalytic domain (TBD) adopting a TIM barrel fold and a unique C-terminal helical domain (HD) devoted to tRNA recognition, except for the animal U20-specific Dus2 enzyme.

Engineering an [FeFe]-Hydrogenase: Do Accessory Clusters Influence O Resistance and Catalytic Bias?

[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O.

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.

Structural and functional characterization of the hydrogenase-maturation HydF protein.

[FeFe] hydrogenase (HydA) catalyzes interconversion between 2H and H at an active site composed of a [4Fe-4S] cluster linked to a 2Fe subcluster that harbors CO, CN and azapropanedithiolate (adt) ligands. HydE, HydG and HydF are the maturases specifically involved in the biosynthesis of the 2Fe subcluster. Using ligands synthesized by HydE and HydG, HydF assembles a di-iron precursor of the 2Fe subcluster and transfers it to HydA for maturation.

The anti-sigma factor RsrA responds to oxidative stress by reburying its hydrophobic core.

Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm for sensing oxidative stress centred on the hydrophobic core of a sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor that responds to disulfide stress in the cytoplasm of Actinobacteria.

Chemical assembly of multiple metal cofactors: The heterologously expressed multidomain [FeFe]-hydrogenase from Megasphaera elsdenii.

[FeFe]-hydrogenases are unique and fascinating enzymes catalyzing the reversible reduction of protons into hydrogen. These metalloenzymes display extremely large catalytic reaction rates at very low overpotential values and are, therefore, studied as potential catalysts for bioelectrodes of electrolyzers and fuel cells. Since they contain multiple metal cofactors whose biosynthesis depends on complex protein machineries, their preparation is difficult.

Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin.

Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding.

An extended dsRBD is required for post-transcriptional modification in human tRNAs.

In tRNA, dihydrouridine is a conserved modified base generated by the post-transcriptional reduction of uridine. Formation of dihydrouridine 20, located in the D-loop, is catalyzed by dihydrouridine synthase 2 (Dus2). Human Dus2 (HsDus2) expression is upregulated in lung cancers, offering a growth advantage throughout its ability to interact with components of the translation apparatus and inhibit apoptosis.

From enzyme maturation to synthetic chemistry: the case of hydrogenases.

Water splitting into oxygen and hydrogen is one of the most attractive strategies for storing solar energy and electricity. Because the processes at work are multielectronic, there is a crucial need for efficient and stable catalysts, which in addition have to be cheap for future industrial developments (electrolyzers, photoelectrochemicals, and fuel cells). Specifically for the water/hydrogen interconversion, Nature is an exquisite source of inspiration since this chemistry contributes to the bioenergetic metabolism of a number of living organisms via the activity of fascinating metalloenzymes, the hydrogenases.

Structure of a kinesin-tubulin complex and implications for kinesin motility.

The typical function of kinesins is to transport cargo along microtubules. Binding of ATP to microtubule-attached motile kinesins leads to cargo displacement. To better understand the nature of the conformational changes that lead to the power stroke that moves a kinesin's load along a microtubule, we determined the X-ray structure of human kinesin-1 bound to αβ-tubulin.

ubiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation.

Coenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI.

Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation.