Projet ANR-06-PCVI-0003 – ERMOE : Engineering the Reactivity of Complex Molybdoenzymes
Programme : Physique et Chimie du Vivant (PCV) (PCV) 2006
Année de financement : 2006
Coordinateur du projet : CNRS DELEGATION REGIONALE PROVENCE ET CORSE (CNRS DELEGATION REGIONALE PROVENCE ET CORSE)
Partenaires : Centre national de la recherche scientifique; Centre national de la recherche scientifique; Commissariat à l'énergie atomique et aux énergies alternatives
Montant : 450000.0 euros
Date de début : 2006
Durée : 36 mois
Résumé : 1- Scientific backgrounds and objectives: Molybdoenzymes (MOEs) are a large class of oxidoreductases which occur in all kingdoms. Being involved in many essential redox processes, ranging from bacterial respiration to the biosynthesis of hormones in higher eukaryotes, they are studied in various contexts, including medical and environmental. Within MOEs, the subclass called "DMSO reductase family" collects bacterial enzymes which use a Mo-bis(molybdopterin) active site to specifically transform small inorganic substrates, often in an oxo-transfer reaction coupled to the transfer of 2 electrons and 2 protons. Some of these substrates (arsenite, chlorate, tellurite and selenate...) are toxic anions whose abundance in both natural and humanly impacted environments is an important issue of current public health. These enzymes also catalyze important reactions in the global carbon, sulphur and nitrogen cycles. The enzymes of the DMSO reductase family are built by assembling conserved redox modules, and their heterogeneity in terms of cofactor contents and quaternary architectures mirrors the diversity of the reactions they catalyze. Their function therefore proves adaptable, but the molecular basis of their reactivity in terms of efficiency, specificity and catalytic directionality is not understood yet. Our aim is to identify the structural determinants which govern the catalytic properties in this family of MOEs by using an integrated strategy that will combine the various techniques of molecular biology, biophysics and modelling.  We will apply this strategy to study a small number of readily tractable model enzymes. We will control their reactivities by modifying the factors that we will have identified. In the long run, our goal is to use genetic engineering on nitrate reductases to enhance their bioremediation activity. 2- Description of the project, methodology : As model systems, we have selected two nitrate reductases and one arsenite oxidase on the following criteria: (1) These three enzymes illustrate the diversity of reactivities, active site coordinations and quaternary structures in this family of enzymes. (2) The reactions they catalyze are related to environmental concerns. (3) Their 3D structures are known (two of them have actually been determined in teams that are directly involved in the project). (4) We are able to produce and purify the proteins in amounts compatible with material-demanding techniques (5) and to selectively modify them by using genetic engineering. We will study these enzymes in an interdisciplinary approach combining biochemistry and site-directed mutagenesis with modern electrochemical techniques, advanced or time-resolved optical and magnetic spectroscopies (EPR, ENDOR, ESEEM, MCD...) and X-ray crystallography. We will focus on all aspects related to function: on the structural, electronic and redox properties of the active sites in these enzymes, on the dynamics of long-range, intramolecular electron and proton transfers, and on the intermolecular steps at the sites of interaction with quinones. Beyond the detailed study of each system, our original strategy will consist in the comparison of the distinct properties of these similar enzymes. Our constant goal will be to tackle transverse and general questions related to the specificity of these enzymes towards their substrates, to the intrinsic directionality of the catalysts, and to the dynamical and integrated aspects of the mechanism. 3- Expected results : In order to test the knowledge we will have acquired on the structural determinants of the reactivity of these complex oxidoreductases, we will use site-directed mutagenesis to divert the catalytic mechanism of a nitrate reductase, with the aim of making it able to work in the opposite (oxidizing) direction, or to increase its reactivity towards alternative substrates, such as toxic oxi-anions. The fact that all the enzymes that have a specific and physiological bioremediation activity