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Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution.

Zhan T, Zhang K, Chen Y, Lin Y, Wu G, Zhang L, Yao P, Shao Z, Liu Z - PLoS ONE (2013)

Bottom Line: Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling.Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities.These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, P. R. China.

ABSTRACT
Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg(51) mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

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A. Docking analysis of glyphosate-B3S1 complex. The atoms of the nine amino acid mutations are shown with stick representation. The flavin cofactor is in yellow and the ligand glyphosate is shown with ball-and-stick representation. B. The model of variant B3S1 active site docking with the substrate glyphosate. The partial accessible space of the active site is shown in green and purple. The main active residues are shown with stick representation, and hydrogen bonds are represented in yellowdottedlines. C. 2D depiction of the glyphosate-residues interaction in variant B3S1. The schematic representation was generated using MOE and the residues are shown in purpledisks. The hydrogen bonds are represented in greendottedlines with the arrow denoting the direction of the bond. The solvent-exposed surface of catalytic residues is drawn as a halo-likedisk around the residue. The solvent exposure of ligand is expressed in contourdottedline, and the solvent exposure of substitution group is shown in bluesmudge.
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pone-0079175-g003: A. Docking analysis of glyphosate-B3S1 complex. The atoms of the nine amino acid mutations are shown with stick representation. The flavin cofactor is in yellow and the ligand glyphosate is shown with ball-and-stick representation. B. The model of variant B3S1 active site docking with the substrate glyphosate. The partial accessible space of the active site is shown in green and purple. The main active residues are shown with stick representation, and hydrogen bonds are represented in yellowdottedlines. C. 2D depiction of the glyphosate-residues interaction in variant B3S1. The schematic representation was generated using MOE and the residues are shown in purpledisks. The hydrogen bonds are represented in greendottedlines with the arrow denoting the direction of the bond. The solvent-exposed surface of catalytic residues is drawn as a halo-likedisk around the residue. The solvent exposure of ligand is expressed in contourdottedline, and the solvent exposure of substitution group is shown in bluesmudge.

Mentions: To identify the possible molecular basis for the enhancement of oxidase activity against glyphosate, we constructed a docking model of the B3S1-glyphosate complex based on the homology model (Figure 3A). Combined with the data of secondary structure predicted by the PSIPRED server [37], we found that two of the valuable mutations introduced into G51R/D60S were located on the loop connecting α2-α3 helix, and Arg51 was close to the active site, which established an electrostatic interaction and hydrogen bonds with the phosphonate group of glyphosate (Figure 3C). On the one hand, the guanidinium group of Arg51 contributed to the stabilization of glyphosate binding, which might enhance the affinity for glyphosate, but decrease the affinity for glycine. On the other hand, this polar residue was prone to provide partially positive charge to neutralize negative charge in the active site, thus increasing the cofactor’s redox potential [38]. The substitution of D60G was generated in evolved mutant 23B1 by replacing an acidic residue with a neutral residue without side chain, which contribute to the improved catalytic activity of GO, mainly because the loop connecting α2-α3 helix could possess a high mobility and bring a corresponding slight conformation change in the proximity of active site [14]. The mutations (G51R/D60G) close to the active site improved the catalytic efficiency of BceGO on glyphosate mainly by decreasing the Km value (up to 35-fold in mutant B1R).


Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution.

Zhan T, Zhang K, Chen Y, Lin Y, Wu G, Zhang L, Yao P, Shao Z, Liu Z - PLoS ONE (2013)

A. Docking analysis of glyphosate-B3S1 complex. The atoms of the nine amino acid mutations are shown with stick representation. The flavin cofactor is in yellow and the ligand glyphosate is shown with ball-and-stick representation. B. The model of variant B3S1 active site docking with the substrate glyphosate. The partial accessible space of the active site is shown in green and purple. The main active residues are shown with stick representation, and hydrogen bonds are represented in yellowdottedlines. C. 2D depiction of the glyphosate-residues interaction in variant B3S1. The schematic representation was generated using MOE and the residues are shown in purpledisks. The hydrogen bonds are represented in greendottedlines with the arrow denoting the direction of the bond. The solvent-exposed surface of catalytic residues is drawn as a halo-likedisk around the residue. The solvent exposure of ligand is expressed in contourdottedline, and the solvent exposure of substitution group is shown in bluesmudge.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818420&req=5

pone-0079175-g003: A. Docking analysis of glyphosate-B3S1 complex. The atoms of the nine amino acid mutations are shown with stick representation. The flavin cofactor is in yellow and the ligand glyphosate is shown with ball-and-stick representation. B. The model of variant B3S1 active site docking with the substrate glyphosate. The partial accessible space of the active site is shown in green and purple. The main active residues are shown with stick representation, and hydrogen bonds are represented in yellowdottedlines. C. 2D depiction of the glyphosate-residues interaction in variant B3S1. The schematic representation was generated using MOE and the residues are shown in purpledisks. The hydrogen bonds are represented in greendottedlines with the arrow denoting the direction of the bond. The solvent-exposed surface of catalytic residues is drawn as a halo-likedisk around the residue. The solvent exposure of ligand is expressed in contourdottedline, and the solvent exposure of substitution group is shown in bluesmudge.
Mentions: To identify the possible molecular basis for the enhancement of oxidase activity against glyphosate, we constructed a docking model of the B3S1-glyphosate complex based on the homology model (Figure 3A). Combined with the data of secondary structure predicted by the PSIPRED server [37], we found that two of the valuable mutations introduced into G51R/D60S were located on the loop connecting α2-α3 helix, and Arg51 was close to the active site, which established an electrostatic interaction and hydrogen bonds with the phosphonate group of glyphosate (Figure 3C). On the one hand, the guanidinium group of Arg51 contributed to the stabilization of glyphosate binding, which might enhance the affinity for glyphosate, but decrease the affinity for glycine. On the other hand, this polar residue was prone to provide partially positive charge to neutralize negative charge in the active site, thus increasing the cofactor’s redox potential [38]. The substitution of D60G was generated in evolved mutant 23B1 by replacing an acidic residue with a neutral residue without side chain, which contribute to the improved catalytic activity of GO, mainly because the loop connecting α2-α3 helix could possess a high mobility and bring a corresponding slight conformation change in the proximity of active site [14]. The mutations (G51R/D60G) close to the active site improved the catalytic efficiency of BceGO on glyphosate mainly by decreasing the Km value (up to 35-fold in mutant B1R).

Bottom Line: Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling.Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities.These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, P. R. China.

ABSTRACT
Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg(51) mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

Show MeSH
Related in: MedlinePlus