Description du sujet de thèse

Scientific background:
Aromatic nitration is a key chemical reaction in organic synthesis. It involves introducing a nitro group (-NO₂) onto an aromatic ring. This process plays a fundamental role in the pharmaceutical industry, which exploits nitration for the synthesis of numerous active ingredients, such as antibiotics and analgesics. However, conventional nitration processes, using mixtures of nitric and sulphuric acids, suffer from major limitations in terms of selectivity, safety and environmental impact.
Faced with these challenges, the emergence of biocatalysis as an alternative to traditional chemical methods represents a promising step forward. The use of nitrating enzymes would enable nitration to be carried out under milder reaction conditions, reducing the formation of undesirable by-products and improving the selectivity of the reaction. This approach would also eliminate the use of strong acids and toxic solvents, thereby reducing the safety risks for operators and the ecological footprint of the process, in full compliance with European REACh regulations.
Our approach is based on enzymatic engineering to develop an optimised protein for the synthesis of 4-nitroaniline, a key intermediate in the pharmaceutical industry. Indeed, 4-nitroaniline is essential for the manufacture of drugs such as paracetamol, dapsone and various antibacterial sulphonamides. Its industrial importance lies in the reactivity of its functional groups, enabling a wide range of chemical transformations necessary for the synthesis of complex compounds.

Objectives:
The aim is to specifically target the cytochrome P450 Thaxtomine E (TxtE) from the bacterium Streptomyces scabiei, whose modification by site-directed mutagenesis could enable the selective nitration of aniline to 4-nitroaniline. Combined computational modelling and site-directed mutagenesis approaches will be used to improve the stereoselectivity and catalytic efficiency of this enzyme. This strategy builds on recent work that has demonstrated the effectiveness of such methods in optimising enzymes for specific chemical transformations, such as the conversion of lovastatin to pravastatin by cytochrome P450 CYP105AS1.

Thesis project and techniques used:

The thesis will focus on the following areas:
(i) Structural and functional characterisation of the TxtE WT enzyme, (ii) Design and in silico optimisation of TxtE mutants , (iii) Experimental validation of the mutants using biochemical tests and crystallography.

- (i) Structural and functional characterisation of the TxtE WT enzyme: In order to establish a structural and functional reference for comparing mutants, the WT enzyme will be produced, purified and characterised using an approach combining biochemistry and biophysics. The first step, consisting of optimising the expression and purification conditions to obtain a homogeneous and stable enzyme, has already been carried out in a preliminary study. From a functional point of view, the enzyme has also been characterised using binding tests, including fluorescence assays, to quantify its affinity for the substrate. A more comprehensive approach will be carried out, including isothermal calorimetry (ITC) and surface plasmon resonance (SPR) methods. A complete kinetic analysis will then enable the Km, kcat and kcat/Km constants to be determined, providing a solid basis for comparison to assess the effect of the mutations introduced during the subsequent stages of the project. Structurally, the aim is to determine the three-dimensional structure of the WT enzyme by crystallisation and X-ray diffraction, which will provide a detailed view of its active site and its potential interactions with the substrate.

- (ii) Design and in silico optimisation of TxtE mutants: Our preliminary results have enabled us to design several mutants using a computational pipeline: mutants were generated using stochastic approaches using the CoulpedMoves algorithm in the Rosetta software, then screened in silico using molecular dynamics to assess the affinity between aniline and each of the mutants (Thermodynamic Integration).

- (iii) Experimental validation of mutants by biochemical tests and crystallography: The most promising mutants, with improved affinity and specificity, will be produced and then evaluated in vitro using the functional tests developed in point 1 (binding and activity tests). The mutants with the best affinity and enzymatic activity will be selected for experimental structure determination.
This approach will be iterative and will alternate in silico (ii) and in vitro (iii) phases in order to guide the engineering of the mutations.

Contexte de travail

The ECMo group of the Tissue Biology and Therapeutic Engineering Laboratory - UMR 5305 CNRS / UCBL specialises in the molecular modelling of small molecules, peptides and proteins in complex environments. The team has 5 permanent members. The PhD student recruited will be assigned full time to this group and will be provided with all the resources required for his or her work (workstation, supercomputer and molecular modelling software). The experimental part will be carried out in the same institute, in the Molecular Microbiology and Structural Biochemistry laboratory - UMR5086 in the Drug Resistance and Membrane Proteins team, which aims to characterise membrane proteins and enzymes of therapeutic interest, using biophysical, enzymology and structural biology (cryo-EM) approaches.
The position is in a sector covered by the protection of scientific and technical potential (PPST), and therefore requires, in accordance with the regulations, that your arrival be authorised by the competent authority of the MESR.

Le poste se situe dans un secteur relevant de la protection du potentiel scientifique et technique (PPST), et nécessite donc, conformément à la réglementation, que votre arrivée soit autorisée par l'autorité compétente du MESR.

Contraintes et risques

No biological or chemical risks