The exploitation of evolutionary information, and more particularly of residue coevolution, has revolutionized protein structure predictions. Adaptation of the methods issued from these analyses to the prediction and design of enzymatic functions remains an open problem. Enzymatic functions are indeed characterized by an internal dynamics of proteins that is difficult to model and study experimentally. In this project, we tackle this problem by studying in detail the capacity of certain hydroxylases of the flavin-containing monooxygenase (FMO) protein family to realize their reaction at different positions of the aromatic cycle of ubiquinone (UQ), a molecule key to the production of cellular energy. More precisely, UQ biosynthesis pathway involves three hydroxylation reactions occurring on three carbons of the UQ aromatic ring. Partner 1 has shown that different proteobacteria species use different combinations of (UQ-)FMOs to hydroxylate these three positions: some bacteria use a single enzyme able to hydroxylate all three positions, while other bacteria use three distinct enzymes that hydroxylate a single position each. The UQ-FMO family is thus characterized by a broad diversity of regioselectivities, with enzymes capable of hydroxylating one, two or three positions of the UQ aromatic cycle. In this context, our objective is to develop a methodology that combines molecular modeling (Partner 2) and evolutionary information of enzymes (Partner 1) to elucidate the molecular mechanisms underlying this diversity of regioselectivities.
Our preliminary results show that in alphaproteobacteria, a family of homologous enzymes named UbiL displays the entire diversity of UQ-FMO regioselectivities, with UbiLs hydroxylating one, two or three positions in different organisms. In addition, an analysis of amino acid coevolution suggests that this diversity is controlled by a sector, i.e., a network of coevolving residues connected in the 3D space (forming a cavity around the active site). In this context, our first objective is to decipher the molecular mechanisms responsible for the variations of the UbiL regioselectivity. To this end, we will use a combination of molecular modeling (Partner 2), of phylogenomics (Partner 1) and of statistical analyses of amino acid coevolution (Partner 1). Moreover, the predicted regioselectivities of natural, artificial and ancestral enzymes (we will resurrect the latter) will be systematically tested using biochemistry experiments (Partner 1). Next, we will apply our methodology to the full set of UQ-FMOs (~1000 sequences) in order to highlight the evolution of mechanisms associated with the hydroxylation stages of the UQ pathway. Finally, we will analyze the entire family of FMOs (~18000 sequences) in order to recapitulate the evolution of this protein family by identifying both commonalities and differences between metabolic pathways. In particular, our objective is to identify the sector(s) responsible for the variations of regioselectivity of UQ-FMOs and, more generally, the variations of regioselectivity of FMOs, and to use this information to refactor enzymatic functions. In this regard, as a proof of concept of the generality of our methodology, we will aim at modifying the regioselectivity of a FMO unrelated to the UQ pathway.
Altogether, this truly interdisciplinary project thus aims at integrating molecular modeling (from 3D modeling of enzymes to the analysis of the internal dynamics of the enzyme in interaction with the substrate) and evolutionary information (from the reconstruction of the evolutionary history of metabolic pathways to the coevolution of amino acids) of enzymes whose functionality can be tested at the bench (using biochemistry experiments) in order to improve our understanding of the functioning and evolution of enzymes, and to propose novel principles for enzyme design.
Monsieur Ivan Junier (Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble)
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.
TIMC-IMAG Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble
LISBP LABORATOIRE D'INGÉNIERIE DES SYSTÈMES BIOLOGIQUES ET DES PROCÉDÉS
Help of the ANR 447,191 euros
Beginning and duration of the scientific project: February 2020 - 42 Months