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.