Open Gate of Corynebacterium glutamicum Threonine Deaminase for Efficient Synthesis of Bulky α-Keto Acids

Abstract

Threonine deaminase (TD, EC 4.3.1.19) mediates α,βelimination (deamination), which has untapped potential in the synthesis of unnatural α-keto acids. However, the narrow substrate scope of wild-type TD limits its application. This issue could be overcome by engineering the substrate tunnel of the enzyme. Here, Corynebacterium glutamicum TD (CgTD) was used as a model, and its substrate tunnel was identified as a gating element. On the basis of the gating characteristics and constitution of the substrate access tunnel of CgTD, an "open-gate" strategy was developed to improve its substrate scope toward bulky substrates. The best variant obtained following this approach, CgTD Mu7 , exhibited 90.6-fold greater catalytic efficiency (k cat / K M ) toward a bulky substrate such as phenylserine. The improvement was due to more efficient substrate coupling, deprotonation, and imine hydrolysis. CgTD Mu7 was used for efficient production of both natural and unnatural α-keto acids, including 2-oxobutyric acid (83.4 g L −1 , 99% conversion), phenylpyruvic acid (72.5 g L −1 , 95% conversion), 2-oxovaleric acid (21.1 g L −1 , 91% conversion), and 2-oxo-4-phenylbutyric acid (29.9 g L −1 , 84% conversion). The proposed strategy provides a theoretical basis for the synthesis of α-keto acids and is also effective for engineering other similar enzymes.

Application Fields, Positions, and Bioinformatic Mining of Non-active Sites: A Mini-Review

“…To expand the scope of threoninedeaminase (TD) to catalyzebulky substrates, Song et aldesigned experiments and found that the TD from Corynebacterium glutamicumcould accommodate a bulky substrate such as phenylserine. They focused on the amino acids located in the substrate tunnel, including the gate constituent residues, anchoring residues, and hinge residues (Song et al, 2020). By the first round of alanine scanning, eight key residues whose activities were ≥30% higher than the wild type(WT) were screened; subsequently, three mutation libraries were constructed by saturation mutation and iterative mutation.…”

“…For substrate catalysis, the prerequisites are as follows: 1) the substrate should fit into the binding pocket of the enzyme, and 2) the substrate should pass through the tunnel (Song et al, 2020).…”

“…In most enzymes, the active site is located in the internal cavity, while the tunnels are connections between the active sites and the solvent (Kokkonen et al, 2019). Therefore, non-active sites located in tunnels are an alternative approach tounderstanding catalytic mechanisms and enzyme engineering (Song et al, 2020). In the tunnel, 47 non-conserved amino acid residues were located; this included 18 gate constituent residues, 12 anchoring residues, and 17 hinge residues which were selected for the expansion of the substrate scope of the enzyme (Song et al, 2020).…”

“…Therefore, non-active sites located in tunnels are an alternative approach tounderstanding catalytic mechanisms and enzyme engineering (Song et al, 2020). In the tunnel, 47 non-conserved amino acid residues were located; this included 18 gate constituent residues, 12 anchoring residues, and 17 hinge residues which were selected for the expansion of the substrate scope of the enzyme (Song et al, 2020). Such an approach has also been applied in other studies (Zhou et al, 2013).…”

Enhanced catalytic efficiency and universality of L-amino acid deaminase achieved by a shorter proton transfer distance

“…Compared to Pmi LAAD, molecular dynamic simulations revealed an increase in root-mean-square fluctuations (RMSFs) for six residues (T105, D144, E145, E340, S412, and E417) around the substrate channel (Figure 5). The increased conformational dynamic of these mutated residues resulted in greater structural flexibility of the channel(W. Song et al 2020;Yang et al 2017), which changed the orientation of the substrate side-chain (phenyl or methylthio group) (Figure 4C and 4E).…”