DOI: 10.5281/zenodo.17493002
title: From Brewing to Plastic Degradation: Uncovering the Polyurethanase Potential of R. chinensis Lipase through Atomistic Simulations

description: Reduced molecular dynamics (MD) trajectories for the open-lid Rhizopus chinensis lipase (RCL) with 4-nitrophenyl butyrate (pNPB)  (3,000 snapshots each, representing 300 ns of GBIS simulation). Reduced MD trajectories for the system containing the apo protein and 40 pNPB molecules placed in the vicinity of the lid and pro-peptide domains  (5,000 snapshots each, representing 500 ns of simulation). Reduced molecular dynamics (MD) trajectories for the quasi-open model chosen for the MD simulations using explicit solvation for RCL with pNPB and 4-nitrophenyl benzylcarbamate (pNC) in both poses Pose A and Pose B  (5,000 snapshots each, representing 500 ns of simulation for 3 replicas for each system). The corresponding starting geometries for all systems mentioned are provided in PDB format. The simulations were performed using the MM level of theory, the AMBER force field, and the NAMD software package.
description: In this computational study, we investigate the potential activity of a lipase produced by Rhizopus chinensis (RCL), a fungus traditionally used in Chinese brewing, to develop enzymatic biodegradation solutions of polyurethane (PUR), one of the most versatile and widely used synthetic polymers. MD simulations in water with low substrate concentrations confirm how the RCL adopts a closed conformation that restricts access to the active site. Upon substrate accumulation or increasing the concentration of substrate, activation of the enzyme is achieved due to conformational changes involving not only the previously proposed rotation of Phe113 but also a significant rearrangement of Arg114. This movement induces the outward shift of Phe113, opening a channel for substrate entry. These findings support the classical interfacial activation mechanism of lipases and justify the selection of a quasi-open lid RCL model for modeling its catalytic activity. Using M06-2X:AM1/MM MD simulations, we validated the esterase activity of RCL on a benchmark ester compound 4-nitrobenzyl butyrate (pNPB), uncovering a standard acylation-hydrolysis mechanism with acylation and hydrolysis steps barriers (18.6 and 19.3 kcal/mol, respectively) in excellent agreement with experimental data. The urethanase activity of RCL was then explored in the degradation of model substrate 4-nitrophenyl benzylcarbamate (pNC). Our results indicate that the process preferentially follows an esterase pathway, fully decomposing the PUR-like model sample through three steps, acylation, hydrolysis, and decarboxylation. The value of the activation energy of the full process, determined by the acylation step (17.2 kcal/mol), indicates a feasible reaction. Comparative analysis between the degradation of pNPB and pNC reveals that RCL's catalytic efficiency is influenced by the geometry and electrostatic nature of the substrate, with the enzyme’s active site aligning key moieties for effective bond cleavage. Additionally, short-range interactions, along with long-range electrostatic effects, polarize key moieties, facilitating charge redistribution during bond formation and cleavage. Our findings provide valuable insights into RCL’s potential as a biocatalyst for PUR degradation and suggest that re-design of the enzyme may include not only mutations to decrease the activation energy of the chemical steps, but also increasing the population of the polymer accessible protein conformations.