Rational surface charge engineering of haloalkane dehalogenase for boosting the enzymatic performance in organic solvent solutions

Abstract

Biocatalysis in organic solvents (OSs) has numerous important applications, but native enzymes in OSs often exhibit limited catalytic performance. Herein, we proposed a computation-aided surface charge engineering strategy to improve the catalytic performance of haloalkane dehalogenase DhaA in OSs based on the energetic analysis of substrate binding to the DhaA surface. Several variants with enhanced OS resistance were obtained by replacing negative charged residues on the surface with positive charged residue (Arg). Particularly, a four-substitution variant E16R/E93R/E121R/E257R exhibited the best catalytic performance (five-fold improvement in OS resistance and seven-fold half-life increase in 40% (vol) dimethylsulfoxide). As a result, the overall catalytic performance of the variant could be at least 26 times higher than the wild-type DhaA. Fluorescence spectroscopy and molecular dynamics simulation studies revealed that the residue substitution mainly enhanced OS resistance from four aspects: (a) improved the overall structural stability, (b) increased the hydrophobicity of the local microenvironment around the catalytic triad, (c) enriched the hydrophobic substrate around the enzyme molecule, and (d) lowered the contact frequency between OS molecules and the catalytic triad. Our findings validate that computation-aided surface charge engineering is an effective and ingenious rational strategy for tailoring enzyme performance in OSs.

1. Introduction

The application of organic solvents (OSs) as reaction media for biocatalysts is indispensable for a broad range of applications in the chemical industries. Enzymatic catalysis in OSs has several advantages, including increased substrate solubility, enhanced selective process, and suppression of unwanted by-product formation [1]. Therefore, non-aqueous biocatalysis in the presence of OSs dramatically lowers the cost of processes [2]. However, the reduced activity, poor stability, and even deactivation of the enzymes by OSs largely limit biocatalytic reactions in neat OSs and aqueous OS mixtures.

Many techniques have been used to explore the interaction between enzymes and OSs from different aspects. For example, conformational changes and structural mobility of enzymes can be experimentally obtained from spectroscopies such as X-ray [3], circular dichroism (CD) [4], or nuclear magnetic resonance [5]. The dynamics of the related solvent shell in aqueous media can be determined adequately by ultrafast fluorescence [6], nuclear Overhauser effect [7], and IR spectra [8]. Furthermore, molecular dynamics (MD) simulations generate a complementary approach to explore the connection between protein dynamics and the stability of enzymes in OSs, which has been proven to exhibit high consistency with numerous experimental measurements [9]. With the help of these techniques, OSs were found to deactivate enzymes mainly via five different mechanisms: (a) conformational changes [10], (b) loss of bound water molecules [11], (c) competitive inhibition [12], (d) interfacial inactivation [13], and (e) thermodynamic stabilization of the substrate ground state [14].

Numerous conventional methods, such as immobilization [15], chemical modification [16], chemical cross-linking [17], and additives [18], have been used to strengthen the stability of enzymes in OSs. In addition, protein-engineering strategies have succeeded in improving the properties of enzymes [18]. As a strategy of protein engineering, protein surface design can not only significantly enhance the stability of enzymes [19] but also change the optimal conditions for enzyme catalysis [20]. Besides, protein surface design has been validated to play an important role in boosting the OS resistance of enzymes [21].