Rational Engineering ofBacillus cereusLeucine Dehydrogenase Towards α-keto Acid Reduction for Improving Unnatural Amino Acid Production

Abstract

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.

One-Pot Biocatalytic Preparation of Enantiopure Unusual α-Amino Acids from α-Hydroxy Acids via a Hydrogen-Borrowing Dual-Enzyme Cascade

“…Then, we introduced NADH-dependent L-leucine dehydrogenase from Bacillus cereus (BcLeuDH) to construct a hydrogen-borrowing dual-enzyme cascade system for catalyzing D-α-HAs to L-UAAs (shown in Figure 1). As in our precious work, we found that BcLeuDH could catalyze not only aromatic substrates but also aliphatic α-keto acids [9,29], which was suitable for this cascade system. Our strategy provided an efficient strategy for biocatalytic preparation of a broad-range L-UAAs, with the enhancement of LbMDH activity towards aromatic and aliphatic α-Has, contributing significantly to the biocatalytic preparation of UAAs.…”

“…With the help of structure and sequence analysis, we had a thorough understanding of the conserved active site and substrate channel of LbMDH. In addition, it has been proved that the residues near the active pocket or substrate channel show a significant effect in enzymatic catalytic efficiency and the substrate affinity [9,32]. Thus, six residues (V127, L189, L193, I204, L243, and Q247) near the active pocket or substrate channel were chosen for site-directed mutagenesis (Figure 2B).…”

“…Unusual amino acids (UAAs) with readily functionalized amine and carboxyl groups are valuable building blocks of modern medicinal chemistry [1][2][3], including free amino acid drugs, anticancer agents, antimicrobial peptides, and cyclic peptide antibiotics, among others [4][5][6][7]. A large amount of drugs and biologically active molecules rely on central UAAs [8,9], such as L-phenylglycine, which has been applied in β-lactam antibiotics including penicillin and pristinamycin I [10,11], while L-α-aminobutyric acid is an important precursor for the synthesis of many chiral drugs like brivaracetam and ethambutol [12,13]. Furthermore, UAAs also have great potential in the preparation of smart nanomaterials, for example, part of dipeptides containing norvaline can be assembled into microporous organic materials [1,14].…”

Improvement of l-Leucine Production in Corynebacterium glutamicum by Altering the Redox Flux

“…Leucine dehydrogenase LeuDH, encoded by leuDH , is a NAD(H)-dependent amino acid dehydrogenase, catalyzing the reductive amination of a variety of aliphatic keto-acids to the corresponding l -amino acids [31]. The reaction is reversible using NAD + as a factor (Figure 1).…”

“…LeuDH, which is an amino acid dehydrogenase using NADH as a cofactor, catalyzes the reversible oxidative deamination and reductive amination between l -leucine or other branched chain amino acids and their corresponding α-keto acids [38]. Although various active or artificial amino acids have been synthesized by leucine dehydrogenases in vitro [6,31], the effect of leucine dehydrogenases in biological l -leucine fermentation has not yet been recognized in detail. Nevertheless, the NAD-dependent LeuDH was expected to be effective for l -leucine biosynthesis.…”