Computational and Enzymatic Analyses Unveil the Catalytic Mechanism of Thermostable Trehalose Synthase and Suggest Strategies for Improved Bioconversion

Abstract

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose to trehalose, and is therefore essential for trehalose production. Consequently, dissecting the catalytic mechanism of TreS is important for enzyme optimization and industrial applications. TreS from Thermobaculum terrenum (TtTreS) is a thermostable enzyme. Here, we studied the composition of the TtTreS active site through computer calculation and enzyme analysis. The results were consistent with a two-step double-displacement mechanism, similar to that of glycoside hydrolase 13 family enzymes. However, our data suggested that glucose rotation, following breakage of the α-1,4 glycosidic bond, is a key factor determining the reaction direction and conversion rate. The N246 residue plays an important role in glucose rotation. Moreover, we established a saturated mutation model for the nonconserved amino acids around the substrate gateway domain. Finally, four TtTreS mutants (K136T, Y137D, K138N, and D139S) resulted in improved trehalose yield compared to that of the wild-type enzyme.

A Novel Trehalose Synthase for the Production of Trehalose and Trehalulose

“…With high substrate specificity and a simple maltose conversion reaction, trehalose synthase has extortionate application for trehalose production. However, its industrial use has been prompted to be limited by poor thermostability and low product yield ( 28 ). Apart from the reversible interconversion of maltose and trehalose, trehalose synthase may act on sucrose, catalyzing the transfer of glucosyl unit onto d -fructose, resulting in the biosynthesis of the sucrose isomer trehalulose.…”

Improving the Synthesis Efficiency of Amino Acids by Analyzing the Key Sites of Intracellular Self-Assembly of Artificial Cellulosome

“…The protein is placed in a square box, the edge of the box is 1.5 nm away from the protein, and 15,000 water molecules are added to solvate the protein. The system was minimized in two stages with constrained and unconstrained optimizations: an initial minimization with the steepest descent (maximum force of 50 kJ/mol/nm in 1500 steps), followed by minimization with the conjugate gradient algorithm (maximum force of 10 kJ/mol/nm in 2000 steps) [32].…”

Analyzing Current Trends and Possible Strategies to Improve Sucrose Isomerases’ Thermostability

“…Precisely, computer-assisted rational design is an attractive alternative that speeds up the process of enzymatic engineering [142]. Commonly, in the in silico stabilization of enzymes, strategies are followed such as comparison with homologous sequences of greater thermostability [143], analysis of the B-factor [144,145], molecular dynamics (MD) simulations [113,146], constraint network analysis (CNA) [147], designing disulfide bonds (DSB) [142,148,149], engineering glycosylation sites [150], designing stabilizing salt bridges based on enzyme sequence and structure [151], and calculations to minimize effective energy that mimics Gibbs free energy [152,153]. During the application of these strategies, substitutions made should not belong to a stabilization center or the active site cavity.…”