Abstract
The inversion from L- to D-stereochemistry endows peptides improved
bioactivity and enhanced resistance to many proteases
and peptidases. To strengthen the
biostability and bioavailability of peptide drugs, enzymatic
epimerization becomes an important way to incorporate D-amino acid into
peptide backbones. Recently, a bifunctional thioesterase NocTE, which is
responsible for the epimerization and hydrolysis of the C-terminal
(p-hydroxyphenyl)glycine residue of β-lactam antibiotic nocardicin A,
exclusively directs to the generation of D-diastereomers. Different from
other epimerases, NocTE exhibits unique stereochemical selectivity.
Herein, we investigated the catalytic mechanism of NocTE via molecular
dynamic (MD) simulations and quantum mechanical/molecular mechanics
(QM/MM) calculations. Through structural analyses, two key water
molecules around the reaction site were found to serve as proton
mediators in epimerization. The structural characteristics inspired us
to propose a substrate-assisted mechanism for the epimerization, where
multi-step proton transfers were mediated by water molecules and
β-lactam ring, and the free energy barrier was calculated to be 20.3
kcal/mol. After that, the hydrolysis of D-configured substrate was
energetically feasible with the energy barrier of 14.3 kcal/mol. As a
comparison, the energy barrier for the direct hydrolysis of
L-configured
substrate was obtained to be 24.0 kcal/mol. Our study provides
mechanistic insights into catalytic activities of bifunctional
thioesterase NocTE, uncovers more clues to the molecular basis for
stereochemical selectivity and paves the way for the directed
biosynthesis of novel peptide drugs with various stereostructural
characteristics by enzyme rational design.