Mechanism‐Guided Computational Design of ω‐Transaminase by Reprograming of High‐Energy‐Barrier Steps

Abstract

ω-Transaminases (ω-TAs) show considerable potential for the synthesis of chiral amines. However, their low catalytic efficiency towards bulky substrates limits their application, and complicated catalytic mechanisms prevent precise enzyme design. Herein, we address this challenge using a mechanism-guided computational enzyme design strategy by reprograming the transition and ground states in key reaction steps. The common features among the three high-energy-barrier steps responsible for the low catalytic efficiency were revealed using quantum mechanics (QM). Five key residues were simultaneously tailored to stabilize the rate-limiting transition state with the aid of the Rosetta design. The 14 top-ranked variants showed 16.9-143fold improved catalytic activity. The catalytic efficiency of the best variant, M9 (Q25F/M60W/W64F/I266A), was significantly increased, with a 1660-fold increase in k cat / K m and a 1.5-26.8-fold increase in turnover number (TON) towards various indanone derivatives.

Stereoselective amino acid synthesis by synergistic photoredox-pyridoxal radical biocatalysis

“…5; see the supplementary materials for computational details). Consistent with previous computational studies on related PLP-dependent enzymes ( 57 , 58 ), the conversion between the internal aldimine and external aldimine 7 requires a low activation barrier (fig. S16).…”

Reprogramming the Transition States to Enhance C–N Cleavage Efficiency of Rhodococcus opacus l-Amino Acid Oxidase

“…The rate-limiting step in LAAO is similar to the C–N cleavage reaction catalyzed by LAAD because both enzymes are dependent on FAD, and their rate-limiting step involves an energy barrier caused by hydride transfer. However, this differs from C–N cleavage catalyzed by transaminases, such as PLP-dependent ω-transaminase, which has more rate-limiting steps . In our study, based on the catalytic mechanism, the energy barrier was reduced by reprogramming [ TS1 ] and [ TS2 ].…”

“…Following a mutation strategy (i.e., introducing a powerful hydrogen bond interaction between the ligand and critical residue of 3CL Pro ), the enzymatic activity was improved by 8-fold . In addition, enzyme energy barrier engineering has been used to improve the physical and catalytic properties of ω-transaminase (ω-TA) and decarboxylase . For example, the catalytic efficiency for bulky substrates was increased by 1.5–26.8-fold in turnover number by decreasing the energy barrier of three high-energy barrier steps in ω-TA .…”

“…An automated, high-throughput directed evolution platform was developed to engineer polymer-degrading enzymes, increasing the glass transition temperature ( T m ) to 82.5 °C . Intelligent computational methods, such as molecular dynamics (MD), , QM, ,− density functional theory (DFT), , kinetic isotope effect calculations, and machine learning, have been developed to determine rate-limiting steps. For example, computational methods have been used to determine the rate-limiting step of reduction amination, carbene, transamination, and other catalytic reactions.…”

“…Intelligent computational methods, such as molecular dynamics (MD), , QM, ,− density functional theory (DFT), , kinetic isotope effect calculations, and machine learning, have been developed to determine rate-limiting steps. For example, computational methods have been used to determine the rate-limiting step of reduction amination, carbene, transamination, and other catalytic reactions. QM/MM calculations have been used to reveal that both hydride transfer and C–N cleavage steps are rate-limiting for 3,5-DAHDH, an enzyme that catalyzes reductive amination.…”