Rational Engineering of Bacillus cereus Leucine Dehydrogenase Towards α-keto Acid Reduction for Improving Unnatural Amino Acid Production

Unnatural amino acids (UAAs) play a key role in modern medicinal chemistry such as small molecules and peptide-based drugs with fast-growing markets. Low efficiency for natural enzymes including leucine dehydrogenase (LeuDH, EC1.4.1.9) are one major challenge for UAA production. Here, rational engineering of LeuDH from Bacillus cereus with a structure-based design approach is studied. The results achieve higher enzymatic activity and stability toward α-keto acid reduction by improving the hydrophobic and rigidity of enzymatic substrate entrance tunnel. High catalytic efficiency for variant E116V is associated with the presence of more hydrophobic tunnels that allows easy substrate diffusion, which is confirmed in absorbance spectroscopy study. For variant T45M/E116V, melting temperature and half-lives of thermal inactivation at 60 °C is 62.8 °C and 29.2 h, respectively, much higher than 48.4 °C and 3.4 h of wild type. Structural analysis indicates that an additional hydrogen bond in β5 fold is formed in variant T45M, which results in a more rigid β5 fold leading to better stability. Furthermore, asymmetric synthesis of α-amino acids with coenzyme regeneration reveals higher productivities for variant T45M/E116V. This study indicates the importance of substrate entrance tunnel for enzymatic activities and stability, the engineered LeuDH would better serve UAA production. Graphical Low yield rates for natural enzymes including leucine dehydrogenase (LeuDH, EC1.4.1.9) are one major challenge to the production of unnatural amino acids in industrial scale. Here, the catalytic activity and stability of Bc LeuDH toward α-keto acid reduction are remarkably improved via rational engineering of substrate entrance tunnel based on a structure-based design approach. This work will better serve the production of especially unnatural α-amino acids.