Engineering D-Lactate Dehydrogenase from Pediococcus acidilactici for Improved Activity on 2-Hydroxy Acids with Bulky...

Engineering D-lactic acid dehydrogenases for higher activity on various 2-oxo acids is important for the synthesis of 2-hydroxy acids that can be utilized in a wide range of industrial fields including the production of biopolymers, pharmaceuticals, and cosmetic compounds. Although there are many D-lactate dehydrogenases (D-LDH) available from a diverse range of sources, there is a lack of biocatalysts with high activities for 2-oxo acids with large functional group at C₃. In this study, the D-LDH from Pediococcus acidilactici was rationally designed and further engineered by controlling the intermolecular interactions between substrates and the surrounding residues via analysis of the active site structure of D-LDH. As a result, Y51L mutant with the catalytic efficiency on phenylpyruvate of 2200 s^-1 mM^-1 and Y51F mutant on 2-oxobutryate and 3-methyl-2-oxobutyrate of 37.2 and 23.2 s^-1 mM^-1 were found, which were 138-, 8.5-, and 26-fold increases than the wild type on the substrates, respectively. Structural analysis revealed that the distance and the nature of the interactions between the side chain of residue 51 and the substrate C₃ substituent group significantly affected the kinetic parameters. Bioconversion of phenyllactate as a practical example of production of the 2-hydroxy acids was investigated, and the Y51F mutant presented the highest productivity in in vitro conversion of D-PLA.