Stepwise Loop Insertion Strategy For Active Site Remodeling To Generate Novel Enzyme Functions

Synthetic organophosphate compounds and their derivatives are highly toxic due to irreversible inhibit acetyl cholinesterase?AChE?and disrupt neurotransmission in the central nervous system for all vertebrates.The phosphotriesterase?PTE,EC3.1.8.1?within the amidohydrolase superfamily can hydrolyze a broad range of OP compounds,including most OP pesticides and chemical warfare agents?CWAs?.The phosphotriestrerase?PTE?has been recognized an ideal candidate for OP detoxification but it is less stable and will be inactivated at high temperature.Due to the active site provides an optimal microenvironment for specific catalytic reactions,modification on relevant residues around the active center of enzyme would have a direct influence on catalytic properties of enzymes,which might provide an evolutionary pathway for creating new catalytic activities of enzyme.In this study,thermophilic lactonease?GkaP-PLL?with weak phosphotriesterase activity in Geobacillus kaustophilus was selected as a model for molecular design.To evolve and obtain a better PTE enzyme,we have performed the following research work:1.Molecular designing to transform a Thermostable GkaP-PLL lactonase into a PTE enzyme.The PTE and a PTE-like lactonase?PLL?belong to the amidohydrolase superfamily,sharing a common??/??₈ TIM-barrel structural fold.GkaP-PLL exhibits a similar folded structure as pdPTE but possesses different active loop configurations.Notably,loop7 of Gka P-PLL is 11amino acids shorter than that of pdPTE.The PTE loop7 has been proposed to contain two possible individual insertion sites,annotated L7-A and L7-B,connected by a 4-amino-acid spacer.There are two more residues in pdPTE L7-A and nine more in L7-Bthan there are in GkaP-PLL loop7.In addition,there are significant differences in the amino acid constitution and spatial architecture.To efficiently elongate GkaP-PLL loop7,we developed stepwise loop insertion strategy?StLois?strategy to design and construct smart mutant libraries that introduced two residues in a stepwise manner with degenerate NNK codon randomization.The residues were gradually introduced into the GkaP-PLL L7-A region by saturation mutagenesis.This method can effectively accumulate the active site loop mutation adaptability,to optimize the mutant enzyme of the substrate specificity.2.Use StLois strategy to construct a series of small intelligent mutant libraries and screening PTE activity.To obtain a more efficient OP hydrolase,we used wild-type GkaP-PLL and our GkaP-PLL variantML7?26A8,F28I/Y99L/T171S/F228L/N269S/V270G/G273D?,whichhas approximately 38 times higher activity than the WT,as templates for loop7 insertion mutagenesis.To avoid a massive,time-consuming library screen,we propose that a saturated mutagenesis library of double residue insertions in the loop would be suitable for each round and that the mutational fitness effect could be compounded to identify stable variants with desired functions.The algorithm P_i=1-?1-F_i?^T was used to count the number of transformants for double?3.0×10³?,triple?9.8×10⁴?and quadruple?3.1×10⁶?residue insertions/deletions/mutations as a function of NNK codon degeneracy with 95%coverage.3.Characterization of evolved variantsBy inserting six residues into active site loop 7,the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency(k_cat/K_m=7.9×10⁴ M^-1s^-1)toward ethyl-paraoxon compared with its initial template ML7 and 609-fold higher than wild type.The best variant shifts substrate specificity>10⁷-fold.The catalytic efficiency of OP hydrolysis has been determined for all of the variants at 37°C.In addition,all of the obtained variants exhibited significantly higher hydrolytic activities for the tested pesticides?from 17-to 1252-fold compared to the WT?.All of the variants showed a higher activity for ethyl-substituted OP derivatives,including ethyl-paraoxon,ethyl parathion,and diazinon,with the exception of the OP chlorpyrifos.4.Structural and molecular dynamic analysis of wild-type and loop-extended enzyme variantsTo address the mechanism of OP hydrolysis with the novel catalysts,we attempted to solve the crystal structures of several variants for structural analysis.However,only variant L7-A2B2?DR-MI insertion?evolved from wild type template,was successfully crystallized and determined at a resolution of 1.9??PDB code:5CH9?.The variant adopts a fold similar to that of the WT,but striking difference a used by the amino acid insertion in loop7 is evident in the active site pocket.The width of the main entrance to the active site,as measured from the D73 of loop2 to A105 of loop3,was 9.4?in the L7-A2B2 variant but only 2.7?in the WT,indicating an enlargement of the active site entrance caused by the additional loop residues.The pocket volume for substrate binding in L7-A2B2 is 1124?³ as measured by CASTp online,which is larger than that in WT?430?³?and might be more suitable for larger OP substrate catalysis.We also performed 80 ns MD simulations on WT and L7-A2B2 mutant complexes.The binding free energy of L7-A2B2 was-28.5±1.3 kcal/mol,which is 42.3%lower than that of the WT?-20.1±0.8 kcal/mol?.The RMSD and RMSF analysis suggest that the mutant L7-A2B2 is more stable with ethyl paraoxon compared to?-decanolactone.Generally,this study demonstrates that StLois is a powerful method for active site remodeling to alter enzyme substrate preference and create new catalysts.The successful change in enzyme function without disrupting the original protein structure has deepened our understanding of the factors that govern molecular evolution in nature.Moreover,this methodology can be readily generalized to design and create novel biocatalysts and even novel functional proteins to perform a wide range of chemical or physiological reactions for which natural enzymes/proteins do not exist.