Abstract
Transaminases that promote the amination of ketones into amines are an emerging class of biocatalysts for preparing a series of drugs and their intermediates. One of the main limitations of (R)-selective amine transaminase from Aspergillus terreus (At-ATA) is its weak thermostability, with a half-life (t1/2) of only 6.9 min at 40°C. To improve its thermostability, four important residue sites (E133, D224, E253, and E262) located on the surface of At-ATA were identified using the enzyme thermal stability system (ETSS). Subsequently, 13 mutants (E133A, E133H, E133K, E133R, E133Q, D224A, D224H, D224K, D224R, E253A, E253H, E253K, and E262A) were constructed by site-directed mutagenesis according to the principle of turning the residues into opposite charged ones. Among them, three substitutions, E133Q, D224K, and E253A, displayed higher thermal stability than the wild-type enzyme. Molecular dynamics simulations indicated that these three mutations limited the random vibration amplitude in the two α-helix regions of 130–135 and 148–158, thereby increasing the rigidity of the protein. Compared to the wild-type, the best mutant, D224K, showed improved thermostability with a 4.23-fold increase in t1/2 at 40°C, and 6.08°C increase in . Exploring the three-dimensional structure of D224K at the atomic level, three strong hydrogen bonds were added to form a special “claw structure” of the α-helix 8, and the residues located at 151–156 also stabilized the α-helix 9 by interacting with each other alternately.
Highlights
Chiral amines are important components of many significant bioactive compounds, pharmaceutical intermediates and agrochemical industry products (Bornscheuer et al, 2012; Mathew and Yun, 2012a; Ghislieri and Turner, 2014; Park et al, 2014; Fuchs et al, 2015; Ferrandi and Monti, 2017; Dawood et al, 2018; Cai et al, 2020)
To predict the residue with an optimized value and analyze the flexibility of the surrounding amino acid residues, ETSS was used to evaluate the interaction of charged amino acids (Zhang et al, 2014)
The program was used to calculate the interaction parameters of At-amine transaminases (ATAs), and the total Eij shown in Figure 1 reveals that there are 95 charged amino acids in the monomer of At-ATA
Summary
Chiral amines are important components of many significant bioactive compounds, pharmaceutical intermediates and agrochemical industry products (Bornscheuer et al, 2012; Mathew and Yun, 2012a; Ghislieri and Turner, 2014; Park et al, 2014; Fuchs et al, 2015; Ferrandi and Monti, 2017; Dawood et al, 2018; Cai et al, 2020). In addition to optical rotation, enantiomers of chiral drugs have the same physical properties, but they are absorbed, activated or degraded by the metabolic system of the human body in different ways during the pharmacological action, Thermostability of the At-ATA resulting in different efficacies and toxicities (Burke and Henderson, 2002). Because of their broad-spectrum biological activities and high purity in the synthesis of enantiomeric amines, they can act as chiral building blocks for the synthesis of more complex structural drugs (Breuer et al, 2004; Nugent, 2010). ATAs can achieve continuous flow biotransformations under mild conditions (Andrade et al, 2014) as an alternative technology to replace toxic, non-recyclable chemical catalysts and reduce the use of high- temperature and high-pressure conditions in the chemical production process
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