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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Published on: July 25, 2013
Semirational Design Based on Consensus Sequences to Balance the Enzyme Activity-Stability Trade-Off
Yang Zhao1,2, Kun Chen1, Haixia Yang1
1National Engineering Research Center for Fruit & Vegetable Processing, Key Laboratory of Fruit & Vegetable Processing, Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-Thermal Processing, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Abstract:
In this study, the phenomenon of the stability-activity trade-off, which is increasingly recognized in enzyme engineering, was explored. Typically, enhanced stability in enzymes correlates with diminished activity. Utilizing Rosa roxburghii copper-zinc superoxide dismutase (RrCuZnSOD) as a model, single-site mutations were introduced based on a semirational design derived from consensus sequences. The initial set of mutants was selected based on activity, followed by combinatorial mutation. This approach yielded two double-site mutants, D25/A115T (18,688 ± 206 U/mg) and A115T/S135P (18,095 ± 1556 U/mg), exhibiting superior enzymatic properties due to additive and synergistic effects. These mutants demonstrated increased half-lives (T1/2) at 80 °C by 1.2- and 1.6-fold, respectively, and their melting temperatures (Tm) rose by 3.4 and 2.5 °C, respectively, without any loss in activity relative to the wild type. Via an integration of structural analysis and molecular dynamics simulations, we elucidated the underlying mechanism facilitating the concurrent enhancement of both thermostability and enzymatic activity.
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