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...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 computationaided surface charge engineering is an effective and ingenious rational strategy for tailoring enzyme performance in OSs.展开更多
The haloalkane dehalogenase LinB from Sphingomonas paucimobills UT26 was found to transform the 1,2,3-trichloropropane（TCP） into inorganic halide ions and 2,3-dichloro-1-propanol although the catalytic activity is v...The haloalkane dehalogenase LinB from Sphingomonas paucimobills UT26 was found to transform the 1,2,3-trichloropropane（TCP） into inorganic halide ions and 2,3-dichloro-1-propanol although the catalytic activity is very low（Kcat=0.005 s^-1）.In this study,molecular dynamics simulation and docking studies were performed to investigate the binding of TCP to LinB.The docking results indicate that LinB does not restrict TCP to be bound productively in the active site and the water-mediated inhibition occurs in the process of TCP interacting with LinB.The residues Ile134,Leu150,Phe154,Pro208,and Ile211 located on the cap domain are potential targets for mutagenesis researches.展开更多
基金funded by the National Key Research and Development Program of China(2018YFA0900702).
文摘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 computationaided surface charge engineering is an effective and ingenious rational strategy for tailoring enzyme performance in OSs.
基金Supported by the National Natural Science Foundation of China(No.20573042)Key Projects in the National Science & Technology Pillar Program of China(No.2006BAE03B01)+1 种基金Specialized Research Fund for the Doctoral Program of Higher Education of China(No.20070183046)Specialized Fund for the Basic Research of Jilin University,China(No.200810018)
文摘The haloalkane dehalogenase LinB from Sphingomonas paucimobills UT26 was found to transform the 1,2,3-trichloropropane（TCP） into inorganic halide ions and 2,3-dichloro-1-propanol although the catalytic activity is very low（Kcat=0.005 s^-1）.In this study,molecular dynamics simulation and docking studies were performed to investigate the binding of TCP to LinB.The docking results indicate that LinB does not restrict TCP to be bound productively in the active site and the water-mediated inhibition occurs in the process of TCP interacting with LinB.The residues Ile134,Leu150,Phe154,Pro208,and Ile211 located on the cap domain are potential targets for mutagenesis researches.
