Rational engineering of the Plasmodium falciparum L-lactate dehydrogenase loop involved in catalytic proton transfer to improve chiral 2-hydroxybutyric acid production

L-lactate dehydrogenases (LDHs) has been widely studied for their ability to reduce 2-keto acids for the produc-tion of 2-hydroxy acids, whereby 2-hydroxybutyric acids (2-HBA) is among the most important fundamental building blocks for synthesizing pharmaceuticals and biodegradable materials. However, LDHs usually show low activity towards 2-keto acids with longer side chain such as 2-oxobutyric acid (2-OBA). Here rational engi-neering of the Plasmodium falciparum LDH loop with residue involved in the catalytic proton transfer was initially studied. By combining homology alignment and structure-based design approach, we found that changing the charge characteristics or hydrogen bond network interactions of this loop could improve enzymatic catalytic ac-tivities and stabilities towards 2-OBA. Compared with wild type, variant N197D(ldh) showed 1.15 times higher ac-tivity and 2.73 times higher K-cat/Km. The half-life of variant N197D(ldh) at 40 degrees C increased to 77.9 h compared with 50.4 h of wild type. Furthermore, asymmetric synthesis of (S)-2-HBA with coenzyme regeneration revealed 95.8 g/L production titer within 12 h for variant N197D(ldh), 2.05 times higher than using wild type. Our study in-dicated the importance of loop with residues involved in the catalytic proton transfer process, and the engineered LDH would be more suitable for (S)-2-HBA production. (C) 2021 Published by Elsevier B.V.