# A Highly Glyphosate-Resistant EPSPS Mutant from Laboratory Evolution
## 3 Results
### Bioinformatic Analysis of GR79(Y40I)-EPSPS  
Clustal alignment of the EPSPS with GR79-EPSPS and some typical Class I, Class II EPSPS (CP4-EPSPS, cb-EPSPS) indicate that the 40 th amino acid Y (tyrosine) of GR79-EPSPS is different from that of other Class I EPSPSs, which are leucine (L), isoleucine (I) or valine (V) (data not shown). Compared with these amino acids, L, I and V are all aliphatic amino acids, while Y is an aromatic amino acid containing a carbon ring. In addition, the amino acid at position 40 of GR79-EPSPS is close to the conserved motif region 1 (GDKX) of Class II EPSPS.  
To investigate the interaction between the glyphosate molecule and the GR79, GR79 (Y40I)-EPSPS proteins, we used molecular dynamics to simulate the molecular dynamics of the protein and GLP molecule complex for 100 ns. The results showed that the RMSD of GR79 and GR79(Y40I)-EPSPS proteins was less than 3.5 A and reached dynamic equilibrium in a short time (20 ns), indicating that the protein itself had good stability (Figure 2A).  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Kinetic Constants** & **GR79-EPSPS** & **GR79(Y40I)-EPSPS** \\ \hline Specific activity (U/mg) & 14.331 \(\pm\) 0.144 & 17.436 \(\pm\) 0.379 \\ IC\({}_{50}\)(glyphosate; mM) & 6.8685 \(\pm\) 1.3785 & 13.995 \(\pm\) 2.815 \\ Km (PEP; uM) & 39.341 \(\pm\) 3.21 & 22.012 \(\pm\) 2.673 \\ Ki (glyphosate; uM) & 75.360 \(\pm\) 5.029 & 127.343 \(\pm\) 14.338 \\ \(V_{\rm max}\)(U/mg) & 9.285 \(\pm\) 0.223 & 21.406 \(\pm\) 0.824 \\ _Ki/Km_ (PEP) & 1.916 & 5.785 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Kinetic parameters of GR79-EPSPS and GR79(Y40I)-EPSPS.  
Figure 1: The growth curves of _E. coli_ harboring pACYC184, pAGR79 and pAGR79(Y40I) plasmids under different concentrations of glyphosate. (**A**) 0 mM glyphosate; (**B**) 200 mM glyphosate; (**C**) 300 mM glyphosate; and (**D**) 400 mM glyphosate. Data are expressed as the average \(\pm\) S.E. of three independent experiments.  
After mutation, the RMSD of the GR79(Y40I)-EPSPS-GLP complex increased significantly, and the binding energy also increased from \(-6.71\) to \(-6.41\) kcal/mol, indicating that the stability of the complex worsened(Figure 2B).  
These results indicate that GLP molecule binding to the enzyme was blocked by the Y40I substitution to improve glyphosate resistance.  
The structure of GR79-EPSPS was modeled in Swiss-model, referring to the _Colwellia psychrythraea_ EPSPS (pdb-5 xwb. 2. A). The structural model of EPSPS shows the position of the Y40I substitution (Figure 3A,B). The protein folded into two globular hemispheric domains connected by a two-stranded hinge. The residue mutated in position 40 was located on the inside of the active site cleft that may form when the two domains are close together. In the GR79(Y40I)-EPSPS mutant, the hydroxyphenyl group of tyrosine was replaced with the methyl group of isoleucine, so both donor and acceptor hydrogen bond counts decreased. Molecular docking results showed that the GLP molecule connected amino acid residues in the active sites (Figure 3C,D), such as Lys-38, Thr-110, Arg-113, and Arg-138, with five hydrogen bonds in the wild-type protein but four hydrogen bonds in the GR79(Y40I)-EPSPS mutant, indicating that the Y40I substitution could weaken competition in the active site with respect to glyphosate. Moreover, the spatial structure of the protein was gently changed in the Y40I substitution; for example, the hydrogen bond distance between the side chain of glyphosate and Lys-38 increased from 2.0 to 2.8 A. We speculate that the decrease in the binding ability of glyphosate to the Y40I mutant was the main reason for the improvement of glyphosate resistance.  
Figure 2: Molecular dynamics analysis diagram. (**A**) Molecular dynamics analysis of GR79-EPSPS and GR79(Y40I)-EPSPS without substrate (glyphosate); (**B**) molecular dynamics analysis of GR79-EPSPS and GR79(Y40I)-EPSPS combined with glyphosate. GR79-EPSPS is marked in dark blue, and GR79(Y40I)-EPSPS is marked in light blue.