Computational Study on the Catalytic Mechanism of UstD Catalyzing the Synthesis of γ‑Hydroxy-α-Amino Acids

The catalytic mechanism of a pyridoxal 5′-phosphate
(PLP)-dependent
UstD was herein studied in atomic detail, employing the computational
hybrid QM/MM methodology. UstD is a PLP-dependent enzyme that catalyzes
the decarboxylative aldol reactions between l-aspartate and
aldehyde or ketone derivatives to form γ-hydroxy-α-amino
acids. In the reaction catalyzed by UstD, the loss of CO2 renders the C–C bond-forming reaction effectively irreversible,
which makes UstD a special case among the enzymes catalyzing the C–C
bond-forming reactions. This enzyme is currently seen as the optimal
approach for the regioselective synthesis of γ-hydroxy-α-amino
acids, which are very difficult to obtain by standard chemical methods.
The results obtained herein showed that the catalytic mechanism of
UstD might follow two paths to occur in three phases: (1) decarboxylation
of substrate l-aspartate, (2) C–C bond formation by
addition of aldehyde, and (3) the regeneration of catalytic sites.
Although Path A and Path B showed a negligible difference in the energy
barrier of the rate-determining step, Path A involves three additional
steps in the overall pathway compared with Path B, which makes the
reaction proceed more readily through Path B. According to the QM/MM
energy profile of Path B, the rate-limiting step of the catalytic
process is the decarboxylation of the side chain of l-aspartate,
which has a calculated energy barrier of 19.19 kcal/mol. Two crucial
residues, H263 and Y257, were identified to interact with the substrate
aspartic acid. The knowledge about the transition states, intermediates,
key residues, and protein conformational changes along the reaction
path will be valuable for engineering UstD to improve the synthesis
of γ-hydroxy-α-amino acids that serve as building blocks
of various high-value chemicals such as antidiabetics and nutritional
supplements.