Reconstitution of Molybdoenzymes with Bis-Molybdopterin Guanine Dinucleotide Cofactors

International audienceMolybdoenzymes are ubiquitous, and play important roles in all kingdoms of life. The enzymes' cofactors comprise the metal molybdenum, a special organic ligand system called molybdopterin (MPT), additional small ligands like water, hydroxide, oxo-, sulfido-or selenido-functions and, in some enzymes, a coordination to the peptide chain of the protein via an amino acid ligand (e.g. serine, aspartate, cysteine or selenosysteine). The so-called molybdenum cofactor (Moco) is deeply buried in the protein at the end of a narrow funnel giving access only to the substrate. In 1974 an assay was developed by Nason and coworkers using the pleotrophic Neurospora crassa mutant nit-1 for the reconstitution of molybdoenzyme activities from crude extracts. These studies lead to the understanding that Moco is the common element in all molybdoenzymes from different organisms. The assay has been further developed since using specific molybdenum enzymes as source of Moco for the reconstitution of diverse purified apo-molybdoenzymes. Alternatively, the molybdenum cofactor can be synthesized in vitro from stable intermediates and can be inserted into apo-molybdoenzymes by the aid of specific Moco-binding chaperones. A general working protocol is described here for the insertion of the bis-molybdopterin guanine dunucleotide cofactor (bis-MGD) into its target molybdoenzyme using the example of Escherichia coli TMAO reductase.

Nitrous oxide production in soil isolates of nitrate-ammonifying bacteria

Here we provide the first demonstration of the potential for N2O production by soil-isolated nitrate-ammonifying bacteria under different C and N availabilities, building on characterizations informed from model strains. The potential for soil-isolated Bacillus sp. and Citrobacter sp. to reduce NO3-, and produce NH4+, NO2- and N2O was examined in batch and continuous (chemostat) cultures under different C-to-NO3- ratios, NO3--limiting (5 mM) and NO3--sufficient (22 mM) conditions. C-to-NO3- ratio had a major influence on the products of nitrate ammonification, with NO2-, rather than NH4+, being the major product at low C-to-NO3- ratios in batch cultures. N2O production was maximum and accompanied by high NO2- production under C-limitation/NO3-sufficiency conditions in chemostat cultures. In media with lower C-to-NO3-N ratios (5- and 10-to-1) up to 2.7% or 5.0% of NO3- was reduced to N2O by Bacillus sp. and Citrobacter sp., respectively, but these reduction efficiencies were only 0.1% or 0.7% at higher C-to-NO3- ratios (25- and 50-to-1). As the highest N2O production did not occur under the same C-to-NO3- conditions as highest NH4+ production we suggest that a re-evaluation may be necessary of the environmental conditions under which nitrate ammonification contributes to N2O emission from soil

A widespread riboswitch candidate that controls bacterial genes involved in molybdenum cofactor and tungsten cofactor metabolism

We have identified a highly conserved RNA motif located upstream of genes encoding molybdate transporters, molybdenum cofactor (Moco) biosynthesis enzymes, and proteins that utilize Moco as a coenzyme. Bioinformatics searches have identified 176 representatives in γ-Proteobacteria, δ-Proteobacteria, Clostridia, Actinobacteria, Deinococcus-Thermus species and DNAs from environmental samples. Using genetic assays, we demonstrate that a Moco RNA in Escherichia coli associated with the Moco biosynthetic operon controls gene expression in response to Moco production. In addition, we provide evidence indicating that this conserved RNA discriminates against closely related analogues of Moco. These results, together with extensive phylogenetic conservation and typical gene control structures near some examples, indicate that representatives of this structured RNA represent a novel class of riboswitches that sense Moco. Furthermore, we identify variants of this RNA that are likely to be triggered by the related tungsten cofactor (Tuco), which carries tungsten in place of molybdenum as the metal constituent

Structural Insights into the Incorporation of the Mo Cofactor into Sulfite Oxidase from Site-Directed Spin Labeling

Mononuclear molybdoenzymes catalyze a broad range of redox reactions and are highly conserved in all kingdoms of life. This study addresses the question of how the Mo cofactor (Moco) is incorporated into the apo form of human sulfite oxidase (hSO) by using site-directed spin labeling to determine intramolecular distances in the nanometer range. Comparative measurements of the holo and apo forms of hSO enabled the localization of the corresponding structural changes, which are localized to a short loop (residues 263–273) of the Moco-containing domain. A flap-like movement of the loop provides access to the Moco binding- pocket in the apo form of the protein and explains the earlier studies on the in vitro reconstitution of apo-hSO with Moco. Remarkably, the loop motif can be found in a variety of structurally similar molybdoenzymes among various organisms, thus suggesting a common mechanism of Moco incorporation

Les Chaperons dédiés à la biogénèse des molybdoenzymes (étude du couple chaperon TorD - molybdoenzyme TorA chez Escherichia coli)

Les molybdoenzymes sont des métalloprotéines dont le site actif est constitué d un cofacteur à molybdène. Ces molybdoenzymes sont retrouvées chez tous les êtres vivants, des bactéries à l homme. Leur biogenèse est un processus complexe qui nécessite la présence de protéines chaperons spécifiques. Au cours de ma thèse, j ai étudié le rôle de la protéine chaperon TorD dans la biogenèse de la molybdoenzyme TorA chez Escherichia coli. TorA est l enzyme terminale périplasmique de la chaîne respiratoire triméthylamine oxyde (TMAO) réductase. J ai montré que le chaperon spécifique TorD, localisé dans le cytoplasme, est essentiel à la protection de la forme non mature de TorA (apoTorA) lors d un stress thermique ou d une carence en cofacteur à molybdène. En effet, l absence de TorD dans ces conditions entraîne la dégradation complète de l apoprotéine. J ai également montré que la séquence signal Tat de TorA, qui permet l export de la protéine vers le périplasme est hypersensible à la dégradation par les protéases. Cette séquence signal pourrait être une voie d entrée pour les protéases qui ensuite dégraderaient l ensemble de l apoenzyme. TorD en interagissant avec la séquence signal de TorA empêche cette première dégradation et permet donc la protection de l apoenzyme. TorD se lie également à la partie globulaire d poTorA. Par cette interaction, TorD permet une maturation optimale de l apoenzyme. Les acides aminés de TorD impliqués dans cette interaction ont été déterminés après mutagenèse aléatoire. Ils sont localisés dans la cinquième hélice de TorD. J ai également montré que TorD présente un rôle de plate-forme sur laquelle se lie le précurseur du cofacteur à molybdène et l enzyme MobA permettant la synthèse de la forme mature du cofacteur. Après catalyse, cette forme mature du cofacteur qui se lie à TorD peut être délivrée à l apoenzyme TorA. Ainsi, TorD connecte tous les éléments nécessaires à la maturation de TorA : d une part il interagit avec le cofacteur à molybdène et d autre part avec l apoenzyme. Nous proposons donc que TorD interagisse à proximité du site actif de TorA pour y délivrer directement le cofacteur à molybdène.T-ALL is a lymphoid neoplasia that accounts for 10-15% of pediatric ALL and 25% of adult ALL. Alarmingly, and despite indisputable success achieved in treatments its incidence is increasing and its prognostic remains pejorative. Survival rate outcome depend notably on a better understanding in pathogenic mechanisms. In this context, the thesis work has been the following: 1) Based on the observation that rare chromosomal SJ keep on recombining in cis using V(D)J recombination, we hypothesized that episomal SJ (ESJ) still remain reactives and can undergo genomic reintegration. We show that mechanistically, ESJ efficiently rearrange in trans and that the cRSS, the sequences targeted in oncogenic chromosomal translocations, are good ESJ integration sites. Moreover, we demonstrate the presence of ESJ reintegration events in vivo and estimate their frequency to ~1/104-6. In conclusion, ESJ reintegration is a potential mechanism of oncogenic deregulation. 2) Conventional and illegitimate V(D)J recombination events (e.g. translocations) are ordered during lymphocyte development. Based on our knowledge on chromosomal translocation mechanisms, we determine the kinetics of a subset of oncogenic activations acquired during the transformation process in a T-ALL patient s leukemic cells. Moreover, we identified up to 10 independent oncogenic events in this patient, illustrating the multi-hit characteristic of T-ALL. Finally, the oncogenic event s functional impact suggests that cMyc play an important role in the particularly aggressive features of the T-ALL developed by this patient.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli

AbstractMolybdenum cofactor (Moco) biosynthesis is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified in bacteria to date. In molybdoenzymes Mo is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into four general steps in bacteria: 1) formation of the cyclic pyranopterin monophosphate, 2) formation of MPT, 3) insertion of molybdenum into molybdopterin to form Moco, and 4) additional modification of Moco with the attachment of GMP or CMP to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on molybdoenzymes, the biosynthesis of Moco, and its incorporation into specific target proteins focusing on Escherichia coli. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems

Bacterial molybdoenzymes: old enzymes for new purposes

International audienceMolybdoenzymes are widespread in eukaryotic and prokaryotic organisms where they play crucial functions in detoxification reactions in the metabolism of humans and bacteria, in nitrate assimilation in plants and in anaerobic respiration in bacteria. To be fully active, these enzymes require complex molybdenum-containing cofactors, which are inserted into the apoenzymes after folding. For almost all the bacterial molybdoenzymes, molybdenum cofactor insertion requires the involvement of specific chaperones. In this review, an overview on the molybdenum cofactor biosynthetic pathway is given together with the role of specific chaperones dedicated for molybdenum cofactor insertion and maturation. Many bacteria are involved in geochemical cycles on earth and therefore have an environmental impact. The roles of molybdoenzymes in bioremediation and for environmental applications are presented

Etude des protéines chaperons de la famille TorD dédiées à la maturation de molybdoenzymes

J'ai décrit au cours de cette thèse une nouvelle famille de protéines chaperons de plus de trente membres impliquées spécifiquement dans la maturation de molybdoenzymes chez les procaryotes. La protéine TorD d'Escherichia coli, notre modèle, est impliquée dans la maturation cytoplasmique de TorA. En effet, TorD interagit directement avec la forme cytoplasmique non mature de TorA (apoTorA). Des études par mutagenèse dirigée laisse penser qu'une région hydrophobe de TorD serait impliquée dans cette interaction. De plus, nous avons montré par des systèmes de reconstitution in vitro que la présence de TorD augmente nettement l'efficacité d'incorporation du cofacteur à molybdène dans apoTorA. TorD induirait un changement conformationnel d'apoTorA qui favorise une conformation apte à acquérir le cofacteur à molybdène. Des études in vivo et in vitro effectuées sur d'autres membres de la famille TorD ont montré que ces chaperons sont spécifiquement dédiés à leur molybdoenzyme partenaire.During my phD, I have described a new chaperone family containing more than thirty members. This family is involved in the maturation of molybdoenzymes in bacteria. The TorD protein of Escherichia coli, our model, is the specific chaperone of periplasmic molybdoenzyme TorA. I have shown that TorD is involved in cytoplasmic maturation of TorA. Indeed, TorD interacts with the cytoplasmic form of TorA (apoTorA). We have defined by directed mutagenesis a hydrophobic patch of TorD involved probably in this interaction. Moreover, I have developed an in vitro system to reconstitute the maturation step of apoTorA. This approach revealed that TorD is essential for a correct molybdenum cofactor insertion in apoTorA. The interaction TorA/TorD modifies the conformation of apoTorA probably to make it competent to receive the molybdenum cofactor. In vivo and in vitro studies on others members of the family showed that these chaperones present a high specificity toward their molybdoenzyme partners.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

The Role of the Nucleotides in the Insertion of the bis-Molybdopterin Guanine Dinucleotide Cofactor into apo-Molybdoenzymes

International audienceThe role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA