Search

Abstract: To improve the thermostability of (R)‐selective amine transaminase from Aspergillus terreus (AT‐ATA), we used computer software Disulfide by Design and Modelling of Disulfide Bonds in Proteins to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilized mutants (N25C‐A28C, R131C‐D134C, M150C‐M280C) from seven candidates by site‐directed mutagenesis. Compared to the wild type, the best two mutants N25C‐A28C and M150C‐M280C showed improved thermal stability with a 3.1‐ and 3.6‐fold increase in half‐life (t1/2) at 40 °C and a 4.6 and 5.1 °C increase in T5010. In addition, the combination of mutant R131C‐D134C and M150C‐M280C displayed the largest shift in thermostability with a 4.6‐fold increase in t1/2 at 40 °C and a 5.5 °C increase in T5010. Molecular dynamics simulation indicated that mutations of N25C‐A28C and M150C‐M280C lowered the overall root mean square deviation for the overall residues at elevated temperature and consequently increased the protein rigidity. The stabilized mutation of R131C‐D134C was in the region of high mobility and on the protein surface, and the disulfide bond constraints the flexibility of loop 121–136. [ABSTRACT FROM AUTHOR]

Glutamate decarboxylase (GAD; EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculation was applied to identify critical amino acid sites affecting thermostability of GAD and select proper mutation contributing to improve structure rigidity of the enzyme. Two mutant enzymes, D203E and S325A, with higher thermostability were obtained, and their semi-inactivation temperature (T5015) values were 2.3 °C and 1.4 °C higher than the corresponding value of the wild-type enzyme (WT), respectively. Moreover, the mutant, S325A, exhibited enhanced activity compared to the wild type, with a 1.67-fold increase. The parallel strategy presented in this work proved to be an efficient tool for the reinforcement of protein thermostability. [ABSTRACT FROM AUTHOR]

To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design and Modelling of Disulfide Bonds in Proteins to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilized mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3.1- and 3.6-fold increase in half-life (t 1/2 ) at 40 °C and a 4.6 and 5.1 °C increase in T 50 10 . In addition, the combination of mutant R131C-D134C and M150C-M280C displayed the largest shift in thermostability with a 4.6-fold increase in t 1/2 at 40 °C and a 5.5 °C increase in T 50 10 . Molecular dynamics simulation indicated that mutations of N25C-A28C and M150C-M280C lowered the overall root mean square deviation for the overall residues at elevated temperature and consequently increased the protein rigidity. The stabilized mutation of R131C-D134C was in the region of high mobility and on the protein surface, and the disulfide bond constraints the flexibility of loop 121-136., (© 2017 International Union of Biochemistry and Molecular Biology, Inc.)