DS10 - Défi des autres savoirs

Understanding and Controlling the Molybdenum Cofactor Reactivity in Enzymes and Maquette Proteins – MOLYERE

Submission summary

Molybdenum and tungsten enzymes are present in most living organisms where they catalyze a wide diversity of reactions. They play essential roles in many metabolic processes of carbon, nitrogen, sulfur and have strong implications in major biogeochemical cycles and in the fields of environment, bioenergies and human health. In most of the prokaryotic enzymes, the Mo or W ion is coordinated by a complex bis-pyranopterin-dithiolene cofactor (Mo/W-bisPGD) and by an amino acid side chain (Ser, Asp, Cys or SeCys). It is astonishing that with a relative minimal set of chemical structures of the Mo/W-bisPGD cofactor, enzymes of this family are the most diverse in terms of reactivity. They catalyze a large range of redox chemical transformations using a broad diversity of substrates including oxides of nitrogen, sulfur, carbon, halogens, and metalloids. However, no correlation appears between the nature of the Mo protein ligand and the catalyzed reactions which emphasizes the crucial role played by the remote surrounding protein to modulate Mo/W cofactor reactivity.
The most striking situation is given by the Mo/W-bisPGD enzyme subfamily with Cys (or Se-Cys) ligand which includes periplasmic nitrate reductases (Nap) and formate dehydrogenases (Fdh). These enzymes share a common Mo/W-bisPGD(S)(S(e)-Cys) cofactor structure with a proximal 4Fe4S cluster, but they catalyze very different chemical processes: an oxygen atom transfer for the nitrate into nitrite reduction (E°’7 = +420 mV) by Nap, and a hydrogen atom transfer for the reversible conversion of formate into CO2 (E°’7 = -430 mV) by Fdh. Because of the potential interest of FdH for catalytic CO2 reduction, these enzymes have attracted much attention. However, despite the availability of several X-ray crystal structures and theoretical calculations giving rise to several hypotheses, the catalytic mechanisms of Nap and Fdh remain very elusive.
The aim of the MOLYERE project is to decipher the molecular factors in the protein environment which control the Mo-cofactor reactivity in the two closely related enzymes Nap and Fdh, and the unique ability of Fdh to reduce CO2. Two original strategies will be conducted in parallel, based both on enzymatic engineering and on the de novo synthesis of artificial Mo-proteins. Our interdisciplinary approach will combine three integrated axes: i) Enzymatic engineering based on detailed phylogenetic analysis to identify and transfer by site-directed mutagenesis the structural factors responsible for the Fdh reactivity towards Nap; ii) The control of the biogenesis of these enzyme to modify and label isotopically either the Mo-bisPGD cofactor or the protein chain. This will be combined with trapping and study of reaction intermediates by advanced EPR and hyperfine spectroscopies, kinetics and theoretical modelling to analyze reaction mechanisms; iii) The de novo synthesis of peptide platforms and artificial proteins to bind the Mo ion in a peculiar molecular environment designed from enzyme studies, in order to study and control its reactivity.
This interdisciplinary project gathers Biologists, Chemists and Physicists of research groups from CNRS, CEA and Aix-Marseille University. These groups are internationally recognized for their strong expertise in the field of molybdoenzymes, genetics and molecular biology, enzyme catalysis, magnetic spectroscopies, peptide synthesis and bioinspired chemistry. Thanks to the iterative exchange of knowledge between the three axes of the project, the determination of the molecular factors controlling the Mo cofactor reactivity will enable to design new bioinspired catalysts for CO2 reduction, a crucial step in CO2 valorization.

Project coordination

Bruno GUIGLIARELLI (Centre National de la Recherche Scientifique Délégation Provence et Corse_ Bioénergétique et Ingénierie des Protéines)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


CEA CADAR-BIAM Institut de biosciences et biotechnologies (BIAM) - Commissariat à l'Energie Atomique et aux énergies alternatives
CNRS DR12_ISM2 Centre National de la Recherche Scientifique Délégation Provence et Corse_Institut des Sciences Moléculaires de Marseille
CNRS DR12_LCB Centre National de la Recherche Scientifique délégation Provence et Corse _Laboratoire de Chimie Bactérienne
CNRS DR12_BIP Centre National de la Recherche Scientifique Délégation Provence et Corse_ Bioénergétique et Ingénierie des Protéines

Help of the ANR 590,332 euros
Beginning and duration of the scientific project: September 2016 - 48 Months

Useful links

Explorez notre base de projets financés



ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter