Speaker
Description
Bacterial quorum sensing (QS) is a cell-to-cell communication with a central role in the virulence of several high-priority pathogenic bacteria, such as Pseudomonas aeruginosa or Acinetobacter baumannii. Degradation of the mediator signal, N-acyl-L-homoserine lactones (AHLs) in Gram-negative bacteria, is an attractive approach to develop alternative antibacterial treatments. Moreover, different pathogens produce different variants of AHLs, which vary on the acyl chain length and/or oxidation state of the C3, which could potentially open the possibility for tailored interventions.
Several natural lactonases can hydrolyze AHLs, although they are usually low-specificity and/or do not possess appropriate biophysical properties such as thermostability, proteolytic resistance or pH tolerance. Even though enzymes possess an enormous structural flexibility that permits to adapt them to new functions and properties, this same flexibility and complexity make their rational redesign highly challenging, even with the recent and continuous advances in experimental and -especially- computational approaches
To overcome these challenges, we developed a computational-experimental framework for enzyme design and optimization integrating biochemical validation experiments into a fully customizable, computational pipeline to generate an iterative refinement process. Starting from AidH, a structurally-known broad-spectrum, non-thermostable lactonase, we computationally generated 15 optimized designs that combine backbone redesign (RFDiffusion3) of the substrate entry pocket with an iteratively optimized sequence (LigandMPNN, AlphaFold3, Rosetta, and PLACER) for the redesigned region biasing substrate preference toward short-chain AHLs, as well as evaluating the substrate stability in the active site using molecular dynamics simulations. Experimentally, we established a bioassay to evaluate the activity of these enzymes and validate it with the wild-type AidH and the catalytically inactive variant AidHS102G. We now aim to biochemically characterize and structurally resolve these variants to obtain a deeper understanding on the structural determinants of substrate specificity to allow rational, substrate-specific redesign of lactonases targeting the AHL profiles of priority pathogens.