Reflections on the Origin of Coded Protein Biosynthesis.

The principle of continuity posits that some central features of primordial biocatalytic mechanisms should still be present in the genetically dependent pathway of protein synthesis, a crucial step in the emergence of life. Key bimolecular reactions of this process are catalyzed by DNA-dependent RNA polymerases, aminoacyl-tRNA synthetases, and ribosomes. Remarkably, none of these biocatalysts contribute chemically active groups to their respective reactions.

Stabilisation of the RirA [4Fe-4S] cluster results in loss of iron-sensing function.

RirA is a global iron regulator in diverse that belongs to the Rrf2 superfamily of transcriptional regulators, which can contain an iron-sulfur (Fe-S) cluster. Under iron-replete conditions, RirA contains a [4Fe-4S] cluster, enabling high-affinity binding to RirA-regulated operator sequences, thereby causing the repression of cellular iron uptake. Under iron deficiency, one of the cluster irons dissociates, generating an unstable [3Fe-4S] form that subsequently degrades to a [2Fe-2S] form and then to apo RirA, resulting in loss of high-affinity DNA-binding.

Reflections on the Origin and Early Evolution of the Genetic Code.

Examination of the genetic code (GeCo) reveals that amino acids coded by (A/U) codons display a large functional spectrum and bind RNA whereas, except for Arg, those coded by (G/C) codons do not. From a stereochemical viewpoint, the clear preference for (A/U)-rich codons to be located at the GeCo half blocks suggests they were specifically determined. Conversely, the overall lower affinity of cognate amino acids for their (G/C)-rich anticodons points to their late arrival to the GeCo.

Structural determinants of DNA recognition by the NO sensor NsrR and related Rrf2-type [FeS]-transcription factors.

Several transcription factors of the Rrf2 family use an iron-sulfur cluster to regulate DNA binding through effectors such as nitric oxide (NO), cellular redox status and iron levels. [4Fe-4S]-NsrR from Streptomyces coelicolor (ScNsrR) modulates expression of three different genes via reaction and complex formation with variable amounts of NO, which results in detoxification of this gas. Here, we report the crystal structure of ScNsrR complexed with an hmpA1 gene operator fragment and compare it with those previously reported for [2Fe-2S]-RsrR/rsrR and apo-IscR/hyA complexes.

Quinolinate Synthase: An Example of the Roles of the Second and Outer Coordination Spheres in Enzyme Catalysis.

The activation energy barrier of biochemical reactions is normally lowered by an enzyme catalyst, which directly helps the weakening of the bond(s) to be broken. In many metalloenzymes, this is a effect. Besides having a direct catalytic action, enzymes can fix their reactive groups and substrates so that they are optimally positioned and also modify the water activity in the system.

The Complex Roles of Adenosine Triphosphate in Bioenergetics.

ATP is generally defined as the "energy currency" of the cell. Its phosphoanhydride P-O bonds are often considered to be "high energy" linkages that release free energy when broken, and its hydrolysis is described as "strongly exergonic". However, breaking bonds cannot release energy and ATP hydrolysis in motor and active transport proteins is not "strongly exergonic".

Oxygen-Sensitive Metalloprotein Structure Determination by Cryo-Electron Microscopy.

Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions.

Nickel and the origin and early evolution of life.

Although nickel (Ni) is a minor element of the Earth's crust, it has played a major role in the evolution of life. This metal is a component of the active sites of several archaeal and bacterial anaerobic enzymes essential for bioenergy processes such as H2 and CO oxidation and CO2 fixation. Furthermore, Ni of meteoritic origin was probably involved in primordial organic phosphorylations.

Transient Formation of a Second Active Site Cavity during Quinolinic Acid Synthesis by NadA.

Quinolinate synthase, also called NadA, is a [4Fe-4S]-containing enzyme that uses what is probably the oldest pathway to generate quinolinic acid (QA), the universal precursor of the biologically essential cofactor nicotinamide adenine dinucleotide (NAD). Its synthesis comprises the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA), which involves dephosphorylation, isomerization, cyclization, and two dehydration steps. The convergence of the three homologous domains of NadA defines a narrow active site that contains a catalytically essential [4Fe-4S] cluster.

Primordial bioenergy sources: The two facets of adenosine triphosphate.

Life requires energy to exist, to reproduce and to survive. Two major hypotheses have been put forward concerning the source of this energy at the very early stages of life evolution: (i) abiotic organics either brought to Earth by comets and/or meteorites, or produced at its atmosphere, and (ii) mineral surface-dependent bioinorganic catalytic reactions. Considering the latter possibility, I propose that, besides being a precursor of nucleic acids, adenosine triphosphate (ATP), which probably was used very early to improve the fidelity of nucleic acid polymerization, played an essential role in the transition between mineral-bound protocells and their free counterparts.

Electron and Proton Transfers Modulate DNA Binding by the Transcription Regulator RsrR.

The [FeS]-RsrR gene transcription regulator senses the redox status in bacteria by modulating DNA binding, while its cluster cycles between +1 and +2 states-only the latter binds DNA. We have previously shown that RsrR can undergo remarkable conformational changes involving a 100° rotation of tryptophan 9 between exposed () and buried () states. Here, we have used the chemical modification of Trp9, site-directed mutagenesis, and crystallographic and computational chemical studies to show that (i) the and states correspond to oxidized and reduced RsrR, respectively, (ii) His33 is protonated in the state due to a change in its p caused by cluster reduction, and (iii) Trp9 rotation is conditioned by the response of its dipole moment to environmental electrostatic changes.

Design of specific inhibitors of quinolinate synthase based on [4Fe-4S] cluster coordination.

Quinolinate synthase (NadA) is a [4Fe-4S] cluster-containing enzyme involved in the formation of quinolinic acid, the precursor of the essential NAD coenzyme. Here, we report the synthesis and activity of derivatives of the first inhibitor of NadA. Using multidisciplinary approaches we have investigated their action mechanism and discovered additional specific inhibitors of this enzyme.

Crystal Structure of the Transcription Regulator RsrR Reveals a [2Fe-2S] Cluster Coordinated by Cys, Glu, and His Residues.

The recently discovered Rrf2 family transcriptional regulator RsrR coordinates a [2Fe-2S] cluster. Remarkably, binding of the protein to RsrR-regulated promoter DNA sequences is switched on and off through the facile cycling of the [2Fe-2S] cluster between +2 and +1 states. Here, we report high resolution crystal structures of the RsrR dimer, revealing that the [2Fe-2S] cluster is asymmetrically coordinated across the RsrR monomer-monomer interface by two Cys residues from one subunit and His and Glu residues from the other.

Crystallographic evidence for unexpected selective tyrosine hydroxylations in an aerated achiral Ru-papain conjugate.

The X-ray structure of an aerated achiral Ru-papain conjugate has revealed the hydroxylation of two tyrosine residues found near the ruthenium ion. The most likely mechanism involves a ruthenium-bound superoxide as the reactive species responsible for the first hydroxylation and the resulting high valent Ru(iv)[double bond, length as m-dash]O species for the second one.

Geochemical Continuity and Catalyst/Cofactor Replacement in the Emergence and Evolution of Life.

The origin of life is mostly divided into "genetics first" and "metabolism first" hypotheses. The former is based on spark-tube tests and organic species from meteorites and comets, and proposes a heterotrophic origin of life also consistent with the "RNA World" concept. The "metabolism first" hypothesis posits that life began autotrophically on minerals and/or hydrothermal vents.

Solar Water Splitting with a Hydrogenase Integrated in Photoelectrochemical Tandem Cells.

Hydrogenases (H ases) are benchmark electrocatalysts for H production, both in biology and (photo)catalysis in vitro. We report the tailoring of a p-type Si photocathode for optimal loading and wiring of H ase through the introduction of a hierarchical inverse opal (IO) TiO interlayer. This proton-reducing Si|IO-TiO |H ase photocathode is capable of driving overall water splitting in combination with a photoanode.

Crystallographic Trapping of Reaction Intermediates in Quinolinic Acid Synthesis by NadA.

NadA is a multifunctional enzyme that condenses dihydroxyacetone phosphate (DHAP) with iminoaspartate (IA) to generate quinolinic acid (QA), the universal precursor of the nicotinamide adenine dinucleotide (NAD(P)) cofactor. Using X-ray crystallography, we have (i) characterized two of the reaction intermediates of QA synthesis using a "pH-shift" approach and a slowly reacting Thermotoga maritima NadA variant and (ii) observed the QA product, resulting from the degradation of an intermediate analogue, bound close to the entrance of a long tunnel leading to the solvent medium. We have also used molecular docking to propose a condensation mechanism between DHAP and IA based on two previously published Pyrococcus horikoshi NadA structures.

The NOX Family of Proteins Is Also Present in Bacteria.

Transmembrane NADPH oxidase (NOX) enzymes have been so far only characterized in eukaryotes. In most of these organisms, they reduce molecular oxygen to superoxide and, depending on the presence of additional domains, are called NOX or dual oxidases (DUOX). Reactive oxygen species (ROS), including superoxide, have been traditionally considered accidental toxic by-products of aerobic metabolism.

Crystal structures of the NO sensor NsrR reveal how its iron-sulfur cluster modulates DNA binding.

NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe-4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other.

Photoelectrochemical H Evolution with a Hydrogenase Immobilized on a TiO-Protected Silicon Electrode.

The combination of enzymes with semiconductors enables the photoelectrochemical characterization of electron-transfer processes at highly active and well-defined catalytic sites on a light-harvesting electrode surface. Herein, we report the integration of a hydrogenase on a TiO-coated p-Si photocathode for the photo-reduction of protons to H. The immobilized hydrogenase exhibits activity on Si attributable to a bifunctional TiO layer, which protects the Si electrode from oxidation and acts as a biocompatible support layer for the productive adsorption of the enzyme.

Crystal Structures of Quinolinate Synthase in Complex with a Substrate Analogue, the Condensation Intermediate, and Substrate-Derived Product.

The enzyme NadA catalyzes the synthesis of quinolinic acid (QA), the precursor of the universal nicotinamide adenine dinucleotide (NAD) cofactor. Here, we report the crystal structures of complexes between the Thermotoga maritima (Tm) NadA K219R/Y107F variant and (i) the first intermediate (W) resulting from the condensation of dihydroxyacetone phosphate (DHAP) with iminoaspartate and (ii) the DHAP analogue and triose-phosphate isomerase inhibitor phosphoglycolohydroxamate (PGH). In addition, using the TmNadA K219R/Y21F variant, we have reacted substrates and obtained a crystalline complex between this protein and the QA product.

A decahaem cytochrome as an electron conduit in protein-enzyme redox processes.

The decahaem cytochrome MtrC from Shewanella oneidensis MR-1 was employed as a protein electron conduit between a porous indium tin oxide electrode and redox enzymes. Using a hydrogenase and a fumarate reductase, MtrC was shown as a suitable and efficient diode to shuttle electrons to and from the electrode with the MtrC redox activity regulating the direction of the enzymatic reactions.

Carbon-sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE.

Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether.

Photoelectrochemical H2 Evolution with a Hydrogenase Immobilized on a TiO2 -Protected Silicon Electrode.

The combination of enzymes with semiconductors enables the photoelectrochemical characterization of electron-transfer processes at highly active and well-defined catalytic sites on a light-harvesting electrode surface. Herein, we report the integration of a hydrogenase on a TiO2 -coated p-Si photocathode for the photo-reduction of protons to H2 . The immobilized hydrogenase exhibits activity on Si attributable to a bifunctional TiO2 layer, which protects the Si electrode from oxidation and acts as a biocompatible support layer for the productive adsorption of the enzyme.