Structural Insights into a Novel Esterase from the East Pacific Rise and Its Improved Thermostability by a Semirational Design

Abstract

Lipolytic enzymes are essential biocatalysts in food processing as well as pharmaceutical and pesticide industries, catalyzing the cleavage of ester bonds in a variety of acyl chain substrates. Here, we report the crystal structure of an esterase from the deep-sea hydrothermal vent of the East Pacific Rise (EprEst). The X-ray structure of EprEst in complex with the ligand, acetate, has been determined at 2.03 Å resolution. The structure reveals a unique spatial arrangement and orientation of the helix cap domain and α/β hydrolase domain, which form a substrate pocket with preference for short-chain acyl groups. Molecular docking analysis further demonstrated that the active site pocket could accommodate p-nitrophenyl (pNP) carboxyl ligands of varying lengths (≤6 C atoms), with pNP-butyrate ester predicted to have the highest binding affinity. Additionally, the semirational design was conducted to improve the thermostability of EprEst by enzyme engineering based on the established structure and multiple sequence alignment. A mutation, K114P, introduced in the hinge region of the esterase, which displayed increased thermostability and enzyme activity. Collectively, the structural and functional data obtained herein could be used as basis for further protein engineering to ultimately expand the scope of industrial applications of marine-derived lipolytic enzymes.

Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

“…5 In addition, the low thermal stability of L-ASNase hinders acrylamide mitigation efficiency in thermally processed food, 6,7 which also increases the risk of microbial contamination and overall production costs. 8 Thus, L-ASNases with desirable thermostability for addressing these shortcomings have always been pursued for the clinical and food industries.…”

“…Due to the short half-life, commercial l -ASNase coming from Escherichia coli (EcA) and Erwinia chrysanthemi (ErA) have a high-dose-dependent disadvantage for ALL treatment, causing severe toxic effects on patients . In addition, the low thermal stability of l -ASNase hinders acrylamide mitigation efficiency in thermally processed food, , which also increases the risk of microbial contamination and overall production costs . Thus, l -ASNases with desirable thermostability for addressing these shortcomings have always been pursued for the clinical and food industries.…”

Engineering the Thermostability of Sucrose Synthase by Reshaping the Subunit Interaction Contributes to Efficient UDP-Glucose Production

“…Stabilizing mutations reduce active-site flexibility, which is required for optimal catalysis. Consequently, increased stability of the mutants usually comes at the expense of catalytic activity. , Although the trade-off problem of stability and activity has been overcome in many thermostable mutants, substantial exploration is required to unlock the intrinsic mechanism of balancing stability and activity. , Future attempts can be made to rationally modify the catalytic pocket of mutant M4 to restore the enzyme’s lost activity, attaining a balance between stability and activity. The interaction among subunits significantly contributed to thermostability improvement, which would not be easily affected by the modification of internal catalytic pockets.…”

“…Proline possesses only one rotatable angle and cannot form the H-bond . It has been suggested that pro-capping the N-terminals of the α-helix/β-sheet can increase the enzyme’s thermostability. − …”

Hot spots-making directed evolution easier

“…The strategy of rigidifying flexible sites has also been applied to many other enzymes including chondroitinase ABC (Kheirollahi et al, 2017), esterase (Zhu et al, 2021), artificial metalloenzymes (Obrecht et al, 2021) and so forth.…”