Rational Molecular Engineering of Glutamate Dehydrogenases for Enhancing Asymmetric Reductive Amination of Bulky α‐Keto Acids

Abstract Glutamate dehydrogenases (GluDHs) are promising biocatalysts for the synthesis of chiral α‐amino acids by asymmetric reductive amination of α‐keto acids. However, their strict substrate specificity limits their applications. To address this problem, we developed a molecular engineering method for GluDHs that enhances the asymmetric reductive amination of bulky α‐keto acids. Based on rational design, a “cave” located in the active site pocket of Pp GluDH (GluDH from Pseudomonas putida ), which plays an essential role in substrate recognition, was tailored to facilitate the accepting of bulky substrates. Two mutants (A167G and V378A) were discovered to have significantly enhanced catalytic activity toward 2‐oxo‐4‐[(hydroxy)(methyl)phosphinyl]butyric acid (PPO) and several other bulky substrates. This molecular engineering method was then applied to ten other GluDHs from different sources and with different properties. All engineered GluDHs acquired substantial improvements in PPO‐oriented catalytic activity. The most efficient mutant of NADP + (nicotinamide adenine dinucleotide phosphate)‐specific GluDHs showed up to 1820‐fold increased activity and the specific activity reached 111.02 U/mg‐protein. The NAD + (nicotinamide adenine dinucleotide)‐specific GluDHs, which have no detectable wild type activity toward PPO, acquired a considerable level of activity (1.90–29.48 U/mg‐protein). In batch production of L‐phosphinothricin, these “cave‐tailored” GluDHs exhibited markedly improved catalytic efficiencies compared with their wild types and ee values of >99%. The space‐time yields (STY) varied from 818.16 to 1482.96 g ⋅ L −1 ⋅ d −1 , suggesting potential practical applications of these mutants. magnified image