Insights into the Catalytic Activity of a Metagenome-Derived Urethanase

The discovery of urethanases shows an opportunity to
access the
biotechnological recycling of polyurethane-based plastics (PURs),
widely used in the manufacture of everyday materials. However, the
mechanistic understanding of these enzymes remains under debate. In
this work, we report a QM/MM-based mechanistic study of the metagenome-derived
urethanase UMG-SP2 catalyzing the degradation of a urethane-like model
compound, 4-nitrophenyl benzylcarbamate (pNC). A high-quality structural
model generated with AlphaFold2, prior to the availability of the
crystal structure, accurately captured the Ser-Ser-Lys catalytic triad
characteristic of amidase signature enzymes. Highly accurate constant-pH
nonequilibrium molecular dynamics and Monte Carlo (neMD/MC) simulations
provided the full titration curve of active site Lys, explaining the
need for alkaline media for the enzyme to be active. The generation
of the free energy landscape, obtained by means of free energy perturbation
methods with the M06-2X DFT functional describing the QM region of
the full system, reveals an esterase-like three-step mechanism of
UMG-SP2, i.e., acylation, hydrolysis, and decarboxylation, with all
steps being kinetically feasible. Our computational results show very
good agreement with experimental kinetic data, with a calculated free
energy barrier of 21.2 kcal·mol–1 for the rate-determining
step compared to 22.9 kcal·mol–1 derived from
the experimentally measured turnover frequency (TOF). The present
results also open the door for the final decarboxylation occurring
in the solution after the release of the product of the hydrolysis
step or within the active site. These findings provide an atomistic
insight into the urethanase function and establish a robust framework
for the future design of biocatalysts targeting polyurethane degradation.