Going Beyond the Local Catalytic Activity Space of Chitinase Using a Simulation-Based Iterative Saturation Mutagenesis Strategy

Abstract

Many advances in modifying enzyme properties have been made based on the computation of stable transition states and experimental directed evolution. However, there are still great challenges related to difficult transition state (TS) calculations and the large number of evolutionary experiments needed for verification. The transition state analogue (TSA) has chemical similarity with the TS structure and can be used as a proxy for the unknown TS. Here, we proposed a computation-driven strategy to optimize structures of enzymes via simulation-based iterative saturation mutagenesis (ISM), aiming to stabilize the spatial effects of the TS with a TSA as a placeholder. This approach was applied to redesign structures of chitinase in order to increase its catalytic activity. After three rounds of simulation-based ISM, the catalytic activity of the triple mutant P55T/S216M/R301T was 29.3-fold higher than that of the wild type. The enrichment ratio of variants with increased activity reached 83% in the library selected for experimental verification. In this study, the strategy of optimizing enzymatic activity by computational ISM based on TSA has been streamlined, which facilitates the process of ISM and can be used to extend the bounds of the local optimal solution space.

Propeptide-Mediated Allosteric Regulation of Xylanase Xyl-1: An Integrated Experimental and Computational Analysis

“…It uses distance and angle to define the active conformation in the conformation set, which facilitates the understanding of the molecular mechanism by which the propeptide affects catalysis. GHs follow the retention mechanism of double displacement, 51 where the carboxylic acid of Glu188 provides a proton to the glycosidic oxygen atom to induce the cleavage of the glycosidic bond. The carboxyl oxygen of Glu97 then nucleophilically attacks the C1 reaction center of Xyl-3 to form a glycosylation intermediate (GE) 52 (Figure 4c).…”

Enhanced Thermostability of an l-Rhamnose Isomerase for d-Allose Synthesis by Computation-Based Rational Redesign of Flexible Regions

“…Considering the potential synergistic interactions between the residues, , these 4 mutants were then combined with M1, resulting in a screening library composed of 10 double-point mutants, 6 triple-point variants, 3 four-point mutations, and 1 five-point mutant. The residual activity of all combined mutants after incubation at 75 °C for 2 h is shown in Figure S3.…”

“…It has been shown that protein engineering strategies such as computer-aided rational or semirational design and directed evolution enable the tailoring of enzymes with improved properties. , The strategy of combining molecular dynamics simulations with design calculations has become a popular approach in recent years . This strategy is amenable to redesigning the structures of enzymes to enhance their catalytic activity with a limited number of mutations, which made a remarkable contribution to the development of synthetic biology . The structure-aided rational design is unlikely to trigger unfolding of protein, and it is increasingly applied to improve the thermostability of enzymes. , The flexible regions often play a significant role in various functions of proteins, including the stability of protein, protein–protein interactions, and substrate and cofactor binding .…”

“…27 This strategy is amenable to redesigning the structures of enzymes to enhance their catalytic activity with a limited number of mutations, which made a remarkable contribution to the development of synthetic biology. 28 The structure-aided rational design is unlikely to trigger unfolding of protein, and it is increasingly applied to improve the thermostability of enzymes. 29,30 The flexible regions often play a significant role in various functions of proteins, including the stability of protein, protein−protein interactions, and substrate and cofactor binding.…”

Enhancing the thermostability and catalytic activity of Bacillus subtilis chitosanase by saturation mutagenesis of Lys242

“…[30] Li enhanced the catalytic activity of Bacillus circulans chitinase through three rounds of saturation mutagenesis, increasing the catalytic activity of the mutant by 29.3-fold. [31] Li et al enhanced the thermostability of Aspergillus niger xylanase by saturation mutating the highly flexible region, resulting in a 137.6fold increase in the half-life of the mutant at 50 • C. [32] Many studies have confirmed that C-terminal mutation is beneficial to improve the catalytic activity and thermostability of the enzyme. [33,34] Xu et al engineered the C-terminal region to improve the thermostability of nitrilase.…”