Biology subjects

Rotilio, L.

Publications and source records attributed to Rotilio, L..

3 recordsLinked to original sources

bioRxiv2026

EnZight: A Structure-Guided Algorithm to Identify and Prioritize Substitution Hotspots for Enzyme Engineering

Homologous protein structures contain valuable information about tolerated sequence variation. However, translating this information into practical enzyme design strategies remains challenging. Here we present EnZight, a user-friendly web server that integrates homologous structural alignment with intuitive visualization to identify substitution hotspots in protein cores. EnZight exploits structurally aligned homologs to identify positions where the surrounding structural environment is conserved while the residue at the position varies across homologs. This enables prediction of substitutions that preserve fold integrity while modulating function and thermostability. The approach further provides interactive structural outputs that allow users to inspect and prioritize substitutions manually. To validate the use of EnZight, we used a polyurethane-degrading amidase as a proof of concept. We constructed 34 variants, and 97% were successfully expressed, indicating high foldability of the predicted substitutions. Several substitutions improved both catalytic turnover and thermostability, and, importantly, beneficial substitutions combined additively, enabling stepwise accumulation of improvements. The best triple mutant variant exhibited a six-fold increase in catalytic turnover and 2{degrees}C increase in apparent melting temperature. Enhanced activity toward the pharmaceutical micropollutant flutamide further demonstrates EnZight's broad applicability in identifying substitutions that enable enzyme optimization across diverse substrates.

bioengineering↗

bioRxiv2026

Fragment Based Active Site Exploration of Urethane Hydrolases Reveals a Diversity of Urethane Binding Modes

Recent advances in the discovery, characterisation, and engineering of urethanases provide new opportunities for the sustainable biocatalytic degradation of polyurethane waste. A mechanistic understanding of enzyme-plastic interactions is essential for structure-based engineering to enhance urethanase activity. However, the extremely complex and hydrophobic nature of polyurethane makes it challenging to elucidate the structural basis of enzyme-plastic interactions. Here, we used a fragment-based approach to characterise the active sites of two novel urethanases with different catalytic scaffolds, employing both a crystallographic fragment-screening (FASE) campaign and soluble fragments of plastic-like analogues that mimic the substrate, transition state, or product. FASE identified new substrate-binding subpockets while interactions of plastic mimetics in the active site provided a mechanistic understanding of the recognition and binding of polyurethane fragments by these subpockets. These results highlight a diversity of binding modes among urethanases toward different polyurethane fragments. SynopsisFragment-based active site exploration of urethanases to elucidate polyurethane binding and cleavage

biochemistry↗

bioRxiv2026

Expanding the Enzymatic Landscape for Polyurethane Degradation of Novel Bacterial Urethanases

Polyurethanes (PURs) represent a significant challenge in plastic waste management due to their chemical resilience and limited recycling options. In this study, we report the identification and characterization of five novel bacterial urethanases, expanding the enzymatic repertoire for targeted PUR depolymerization. These enzymes demonstrated carbamate-cleaving activity optimally under alkaline conditions, maintaining stability across a pH range of 7 to 10 and varying thermal and solvent tolerances. Two candidate enzymes, u17 u15 collectively exhibited high activity, catalytic efficiency, and thermostability, establishing a strong foundation for further optimization. Among them, u15 emerged as particularly notable for its catalytic efficiency on the carbamate model substrate di-urethane ethylene methylenedianiline, DUE-MDA, with a kcat/KM of 51.8 {+/-} 0.1 (s-1mM-1). and this motivated its selection for detailed structural analysis. High-resolution crystallography of u15 revealed key active-site architecture, including the conserved amidase signature catalytic triad and flexible loop regions that influence substrate binding and specificity. Molecular docking and molecular dynamics simulations further elucidated substrate binding determinants of u15 during urethane bond hydrolysis. Docking of DUE-MDA revealed two distinct substrate orientations (Pose A and Pose B) differing in the positioning of the carbamate group relative to Ser177. Pose A was more stable and catalytically competent, maintaining the substrate within the oxyanion hole and sustaining optimal geometry for nucleophilic attack by Ser177. Comparable behavior was observed for the partially hydrolyzed intermediate mono-urethane ethylene methylenedianiline, MUE-MDA, indicating a conserved binding mode across substrates. To further assess enzymatic performance on a realistic industrial material, the panel was then tested on a generic flexible foam substrate derived from 2,4- and 2,6-toluene diisocyanate (TDA), where u15 and u17 emerged as the most active candidates. Collectively, we benchmark the structural framework presented by enzymes in the amidase signature family as a strong foundation for further optimization aiming at advancing sustainable and scalable biocatalytic recycling of polyurethanes.

biochemistry↗