Computational Library Design for Increasing Haloalkane Dehalogenase Stability

Abstract

We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app , 72.5 °C) and an over 200-fold longer half-life at 60 °C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.

X-ray crystallographic validation of structure predictions used in computational design for protein stabilization

“…These results demonstrate that the strategy not to mutate residues close to the active site was successful for the design of fully active, thermostable LEH‐P variants. Similarly, stabilizing mutations in the dehalogenase LinB only reduced enzyme activity when they were introduced close to the active site . This strategy could be a simple but effective way to maintain catalytic activity during computational design, provided that more distant mutations do not reduce activity.…”

“…We have recently explored a strategy termed FRESCO, which uses computational library design, for rapidly increasing the thermostability of enzymes. It was applied to obtain highly thermostable multi‐site mutants of limonene epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, and to obtain thermostable and solvent‐resistant mutants of a haloalkane dehalogenase . LEH catalyzes the enantioconvergent production of (1 S ,2 S ,4 R ) limonene‐1,2‐diol from (4 R )‐limonene‐1,2‐epoxide .…”

“…There is a need to further improve the FRESCO strategy since only one‐third of the mutations predicted to be stabilizing indeed increased thermostability when tested experimentally . A critical step is the prediction of mutant structures, which is relevant at three stages in the design process .…”

Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study

“…The enhanced stability of D311E lipase was due to additional inter-loop interactions, which were indicated by the atomic details of D311E lipase with the observed additional ion pair and hydrogen bond interactions (Ruslan et al, 2012). Mutations located in or contacting flexible regions of the haloalkane dehalogenase LinB had a larger stabilizing effect than mutations outside such regions (Floor et al, 2014). Results of another study demonstrated the enhancement of C. antarctica lipase B kinetic stability by increasing the rigidity of its active site (Xie et al, 2014).…”

FireProt: web server for automated design of thermostable proteins

“…The original protocol was modified to enable fully automated calculation at the reasonable time, while maintaining high prediction accuracy (Supplementary Table S6). Prediction of eight multiple-point mutants using this modified protocol was validated using the data of FRESCO (44) and identified mutations were compared with another online protein stabilization tool PROSS (17). FireProt and PROSS showed similar predictive power, correctly identifying 29 and 20 potentially stabilizing positions, respectively (Supplementary Table S7).…”