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.

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

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

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

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

Bacterial Molybdoenzymes: Chaperones, Assembly and Insertion

International audienceThe biogenesis of molybdoenzymes is a cytoplasmic event requiring both the folded apoenzymes and the matured molybdenum cofactor. The structure and the complexity of the molybdenum cofactor varies in each molybdoenzyme family and consequently different accessory proteins are required for the maturation of the respective enzymes. Thus, for enzymes of both the DMSO reductase and xanthine oxidase families, specific chaperones exist which are dedicated to increase the stability and the folding of specific members of each family. In this review, we describe the role of these chaperones for molybdoenzyme maturation. We present a model which describes step by step the mechanism of the maturation of representative molybdoenzymes from each famil

The Shewanella genus: ubiquitous organisms sustaining and preserving aquatic ecosystems

International audienceThe Gram-negative Shewanella bacterial genus currently includes about 70 species of mostly aquatic γ­-proteobacteria, which were isolated around the globe in a multitude of environments such as surface freshwater and the deepest marine trenches. Their survival in such a wide range of ecological niches is due to their impressive physiological and respiratory versatility. Some strains are among the organisms with the highest number of respiratory systems, depending on a complex and rich metabolic network. Implicated in the recycling of organic and inorganic matter, they are important components of organism-rich oxic/anoxic interfaces, but they also belong to the microflora of a broad group of eukaryotes from metazoans to green algae. Examples of long-term biological interactions like mutualism or pathogeny have been described, although molecular determinants of such symbioses are still poorly understood. Some of these bacteria are key organisms for various biotechnological applications, especially the bioremediation of hydrocarbons and metallic pollutants. The natural ability of these prokaryotes to thrive and detoxify deleterious compounds explains their use in wastewater treatment, their use in energy generation by microbial fuel cells and their importance for resilience of aquatic ecosystems

Protection of the general stress response σ S factor by the CrsR regulator allows a rapid and efficient adaptation of Shewanella oneidensis

International audienceTo cope with environmental stresses, bacteria have evolved various strategies, including the general stress response (GSR). GSR is governed by an alternative transcriptional σ factor named σS (RpoS) that associates with RNA polymerase and controls the expression of numerous genes. Previously, we have reported that posttranslational regulation of σS in the aquatic bacterium Shewanella oneidensis involves the CrsR-CrsA partner-switching regulatory system, but the exact mechanism by which CrsR and CrsA control σS activity is not completely unveiled. Here, using a translational gene fusion, we show that CrsR sequesters and protects σS during the exponential growth phase and thus enables rapid gene activation by σS as soon as the cells enter early stationary phase. We further demonstrate by an in vitro approach that this protection is mediated by the anti-σ domain of CrsR. Structure-based alignments of CsrR orthologs and other anti-σ factors identified a CsrR-specific region characteristic of a new family of anti-σ factors. We found that CrsR is conserved in many aquatic proteobacteria, and most of the time it is associated with CrsA. In conclusion, our results suggest that CsrR-mediated protection of σS during exponential growth enables rapid adaptation of S. oneidensis to changing and stressful growth conditions, and this ability is probably widespread among aquatic proteobacteria