Insight into Improved Thermostability of Cold-Adapted Staphylococcal Lipase by Glycine to Cysteine Mutation.

Jiivittha Veno,Malihe Masomian,Mohd Shukuri Mohamad Ali,Nor Hafizah Ahmad Kamarudin,Raja Noor Zaliha Raja Abd Rahman

doi:10.3390/molecules24173169

Jiivittha Veno, Malihe Masomian + Show 3 more

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https://doi.org/10.3390/molecules24173169

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Abstract

Thermostability remains one of the most desirable traits in many lipases. Numerous studies have revealed promising strategies to improve thermostability and random mutagenesis often leads to unexpected yet interesting findings in engineering stability. Previously, the thermostability of C-terminal truncated cold-adapted lipase from Staphylococcus epidermidis AT2 (rT-M386) was markedly enhanced by directed evolution. The newly evolved mutant, G210C, demonstrated an optimal temperature shift from 25 to 45 °C and stability up to 50 °C. Interestingly, a cysteine residue was randomly introduced on the loop connecting the two lids and accounted for the only cysteine found in the lipase. We further investigated the structural and mechanistic insights that could possibly cause the significant temperature shift. Both rT-M386 and G210C were modeled and simulated at 25 °C and 50 °C. The results clearly portrayed the effect of cysteine substitution primarily on the lid stability. Comparative molecular dynamics simulation analysis revealed that G210C exhibited greater stability than the wild-type at high temperature simulation. The compactness of the G210C lipase structure increased at 50 °C and resulted in enhanced rigidity hence stability. This observation is supported by the improved and stronger non-covalent interactions formed in the protein structure. Our findings suggest that the introduction of a single cysteine residue at the lid region of cold-adapted lipase may result in unexpected increased in thermostability, thus this approach could serve as one of the thermostabilization strategies in engineering lipase stability.

Highlights

Stability plays a potent role in determining the level of protein functionality
To assess the effects of mutation on structural conformation of both wild-type and mutant G210C, we conducted in silico modeling and molecular dynamics (MD) simulation
The plasticity of rT-M386 as a cold-adapted lipase was clearly observed at both temperatures

Summary

Introduction

Stability plays a potent role in determining the level of protein functionality. Higher stability makes a protein more economical as it reduces enzyme turnover and becomes more robust in function, such as in extreme conditions. The term ‘stability’ denotes a protein resistance towards various deleterious factors such as heat or denaturants that could affect its molecular integrity or biological function upon exposure [1]. Many native proteins or enzymes have been studied for their unique. The ability of native enzymes to demonstrate such stability has prompted the search for the underlying factors at molecular and structural levels. Structural adaptation strategies of the extremophiles are unique. Variations in the sequence and structure of psychrophilic enzymes to their higher counterparts (mesophilic and thermophilic enzymes) have significantly revealed the influence of specific trends of amino acids to their adaptation strategies

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