Structural Basis And Rational Design Of The Thermophilic Feruloyl Esterase GthFAE

Feruloyl esterases（FAEs）are indispensable biocatalysts catalyzing the cleavage of ester bonds between polysaccharides and their hydroxycinnamoyl cross-links.The enzymes have variety biotechnological applications as potential biocatalysts in pharmaceutical biosynthesis,food and feed industries,paper production,biomass degradation and production of renewable fuels.FAEs were identified from a variety of sources presenting a broad range of structural,biochemical and enzymatic characteristics,however,seldom of them performed well in alkaline conditions or temperatures higher than 60℃,which might not meet the demand for industrial applications,since,for example,the bleaching stage for paper manufacturing should be conducted in alkaline and70-80℃conditions.Accordingly,it is crucial to develop thermophilic alkaline feruloyl esterase for expanding its industrial applications.Thermostable enzymes are mainly discovered by two ways:identified from thermophilic bacteria and protein engineering.A feruloyl esterase from Geobacillus thermoglucosidasius,Gth FAE,was identified as a thermophilic alkaline feruloyl esterase with potential applications in paper manufacturing.For further improvement of the esterase,the strategy of computer-aided rational design guided by protein structure was attractive,due to its efficiency and cost-saving with an increasing computational tools and structural information available in recent years.The structure of Gth FAE was solved by molecular replacement at 1.9（?）,revealing a core domain of classicalα/βhydrolase fold and an insertedα/βcap domain.The structure belonged to the space group P 1 2₁1,and each asymmetric unit contained two identical molecules resulting from the crystal packing instead of dimerization.The catalytic triad S114-H232-D202 was located between the core and cap domains,with the flexible cap domain regulating the entrance of substrates.The catalytic pocket shaped by the cap domain was open and exposed.A large number of salt bridges in the core domain and comparatively large hydrophobic surface area contributed to its high thermostability and alkaline tolerance.In silico analysis based on the crystal structure helped us in the screening of possible beneficial mutants,and we identified a mutant N142T which could improve the catalytic activity by 136%.The substitution of Asn by Thr resulting in a shorter side chain and a slightly larger catalytic pocket contributed to the improvement.Meanwhile,the rational design of Gth FAE for enhancing thermostability obtained more positive results.The high flexible residues determined by their B-factors were screened both in silico and in experiments.The virtual saturation mutations were conducted on the residues with highest flexibility as the initial screening by Fold X,and then several site-directed mutations were conducted,of which substitutions at residues T41 and T150 apparently improved the thermostability.The T_mof T41N,T150Y and T150R mutants were 2.6℃,3.9℃and 3.6℃higher than the wild type（73.6℃）,respectively,with T150Y highest by 77.5℃.For better performance,the substitutions at the residues were combined.The combination mutant T41N/T150R exhibited an optimal temperature of 65℃,a 6.4℃higher T_mcompared to wild type by 80℃,and a 35-fold longer in half-life（201 min）at70℃.Molecular dynamics simulations helped us to investigate how the residues support the positive effects on the stability.According to the analysis,the structure of T41N/T150R was more stable than the wild type mainly due to the salt bridge and hydrogen bond introduced by T150R with E154 and D164 that stabilized the cap domain.Structural analysis plays a crucial role in studying the structure-function relationship of biological macromolecules and exploring their mechanism of action.However,only a few structures of bacterial FAEs were approachable that our understanding of these esterases was not sufficient.This study not only highlighted on the theoretical foundation and preliminary information of feruloyl esterases with the crystal structure of Gth FAE,but also promoted their industrial development and applications by protein engineering strategies targeted on thermostability.