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 aamino acids by asymmetric reductive amination of a-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 a-keto acids. Based on rational design, a "cave" located in the active site pocket of PpGluDH (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 À1 · d À1 , suggesting potential practical applications of these mutants.

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient l-homophenylalanine synthesis

“…The low degree of freedom of the phenyl group side chain may be the reason for the low k cat value, and thereby, for the low k cat / K m value of the wild-type Bb PheDH toward 2-OPBA. This observation was similar to that in a previous study [ 41 ]. In contrast, the incorporation of three crucial mutations, V309G, L306V, and V144G, gradually enlarged the volume of the substrate-binding pocket compared with that of Bb PheDH.…”

Structure-Guided Steric Hindrance Engineering of Bacillus Badies Phenylalanine Dehydrogenase for Efficient L-Homophenylalanine Synthesis

“…Moreover, the observed enlargement of the substrate-binding pocket can be further supported by calculating the volume of the substrate-binding pocket, which revealed that the volume of the eventual mutant M3-2 was about 196.9 Å 3 larger than that of the wild-type BbPheDH (Additional le 1: Table S5). These results demonstrate that the enlargement of the substrate-binding pocket in a reasonable range was bene cial for the interaction between the enzymes and the bulky substrates [31,33].…”

“…Steric hindrance of the substrate-binding pocket plays a signi cant role in modulating the catalytic properties of an enzyme, including enantioselectivities [24,25], substrate speci cities [26,27], and catalytic activities [25,28,29]. Recent enzyme engineering studies focused on increasing or conferring enzyme activity towards non-native bulky substrates by attenuating the steric hindrance of the substratebinding pocket [30,31]. The transaminase ATA-117 mutant constructed by Merck and Codexis [32] is a remarkable example of such engineering, as the mutant showed marginal reductive amination activity toward the bulky prositagliptin ketone, which was successfully used to catalyze the synthesis of the antidiabetic compound sitagliptin.…”

Transamination-Like Reaction Catalyzed by Leucine Dehydrogenase for Efficient Co-Synthesis of α-Amino Acids and α-Keto Acids

“…However, amino acid dehydrogenase thermodynamically favors the reductive amination reaction of α-keto acids, which uses the expensive coenzyme NADH as a reducing agent [28,29]. This necessitates the coupling of the amino acid dehydrogenase with an additional enzyme such as glucose dehydrogenase (GDH) or formate dehydrogenase (FDH) to regenerate NADH and improve the total turnover number (TTN) [30][31][32][33], which increases the cost and complexity of the process. Thus, to facilitate the simultaneous synthesis of α-amino acids and α-keto acids in a single self-sustaining reaction, we attempted to construct a novel transamination-like reaction catalyzed by an amino acid dehydrogenase by coupling the oxidative deamination of an amino acid and the reductive amination of a keto acid (Figure 1).…”