Directed Evolution Of Glycine Oxidase And Site-saturation Mutagenesis Of F247 Residue

Glyphosate is a widely used broad spectrum herbicide in the world.By blocking the shikimate pathway which is important for aromatic amino acid synthesis in plants,the weeds are removed by glyphosate.However,the normal growth of crops is influenced due to belong to plants,and there will be glyphosate accumulation inside the crops,and then the environment and human health would be damaged.Glyphosate can be degraded by glycine oxidase(GO)through C-N cleavage of the molecular.From this point,the inhibition to crops growth from the herbicide is relieved and the glyphosate residual amount inside the crops will be decreased.Therefore,it is significantly important to the agriculture,the environment and the human health to do the research on glyphosate-degrading gene.In this study,B3S1 was improved with DNA shuffling.A mutant B4S7 was obtained from 10,000 clones.The Km,kcat and kcat/Km values of B4S7 on glyphosate 0.1 mM,3.62 min-1 and 36.2 mM-1 min-1,respectively.The three paramaters on glycine were 50.34 mM,2.18 min-1 and 0.04 mM-1 min-1,respectively.Compared with B3S1,the affinity and catalytic efficiency of B4S7 on glyphosate showed 4.3-fold and 0.6-fold of increase,separately.And to glycine,the affinity and catalytic efficiency all decreased.The specificity constant(kcat/Km ratio between glyphosate and glycine)was 836,which presented a 3.9-fold enhancement than that of B3S1.Especially,the Km value of B4S7 to glyphosate was much less than that of the reported GO(s).In addition,the pH and temperature optima of B4S7 were the same as that of B3S1,and the two enzyme exhibited similar profiles of pH and temperature stabilities.The B4S7 activity was strongly inhibited by Fe2+,but Ca2+、Ba2+、Sr2+ showed activation to the enzyme.Sequence analysis revealed that B4S7 contained four amino acid substitutions(V198I,F247S,S307L and G357E).Structure modeling and molecular docking based on the software MOE demonstrated that the new mutation point F247S located in the vicinity to the active center.In order to study the role of the site,the other 19 amino acids were respectively introduced into the site by site-saturation mutagenesis.The result showed that,compared with B3S1,when phenylalanine was replaced by serine or arginine,the specificity constant of the mutant(F247S and F247R)exhibited a 0.64-fold and 2.01-fold of increase.When mutated to glutamic acid,the specificity constant of the mutant(F247E)decreased 2.1-fold.When replaced by proline,the enzyme inactivated.Therefore,the site played an significant role in regulating substrate specificity.According to the structure model and the previous study about GO,site-directed mutagenesis based on PCR was performed to construct mutant A54R.The Km and kcat/km of A54R on glyphosate were 0.049 mM and 61.02 mM-1 min-1,indicating a 1.04-fold increase of affinity and 0.8-fold of increase of catalytic efficiency compared with B4S7,and a 9.81-fold increase of affinity and 1.89-fold increase of catalytic efficiency compared with B3S1.At the same time,the affinity and catalytic efficiency of A54R to glycine all decreased.The specificity constant of A54R was 1907,presenting a 1.28-fold and 10.26-fold enhancement compared to that of B4S7 and B3S1,respectively.All these observations indicated the substrate specificity of the mutant was changed.This study provides new information for the improvement of GO and the study of structure-function relationship,and could lay the basis for the development of glyphosate-toletant crops.