Crystal structures of herbicide-detoxifying esterase reveal a lid loop affecting substrate binding and activity

Abstract

SulE, an esterase, which detoxifies a variety of sulfonylurea herbicides through de-esterification, provides an attractive approach to remove environmental sulfonylurea herbicides and develop herbicide-tolerant crops. Here, we determined the crystal structures of SulE and an activity improved mutant P44R. Structural analysis revealed that SulE is a dimer with spacious binding pocket accommodating the large sulfonylureas substrate. Particularly, SulE contains a protruding β hairpin with a lid loop covering the active site of the other subunit of the dimer. The lid loop participates in substrate recognition and binding. P44R mutation altered the lid loop flexibility, resulting in the sulfonylurea heterocyclic ring repositioning to a relative stable conformation thus leading to dramatically increased activity. Our work provides important insights into the molecular mechanism of SulE, and establish a solid foundation for further improving the enzyme activity to various sulfonylurea herbicides through rational design.

Fig. 1. Chemical structure of commonly used sulfonylurea herbicides.

The seven herbicides are metsulfuron-methyl, ethametsulfuron-methyl, bensulfuron-methyl, sulfometuron-methyl, thifensulfuron-methyl, tribenuron-methyl and chlorimuron-ethyl, respectively.

Fig. 2. Crystal structure of SulE.

a Overall structure of SulE dimer. The core domain in chain A is shown in cyan. The cap domain and β hairpin are shown in hot pink and yellow, respectively. b Overall structure of SulE monomer from chain A. Secondary structure elements are labeled.

Fig. 3. Structural and mutagenesis analysis of SulE.

a–g The substrate binding pocket of S209A/H333A with MM, EM, TrM, CE, SM, TM, and BM, respectively. Residues (Ile43 and Tyr45) belong to the lid loop of another subunit are shown in light pink. Arg150 is displayed in deep salmon. Other residues involved in substrate binding are shown in white. The seven sulfonylureas are also highlighted in different colors. Hydrogen bonds are shown in black dashed lines. The red dotted line indicates the distance. h Superposition of the seven substrates. i The relative activity of WT SulE and its variants to MM. Data were presented as mean values ± SD, n = 3. Error bars represent the standard deviation from three repeats. ND not detected. Statistical analysis was performed by the two-tailed t-test. *p = 0.0020. Source data are provided as a Source Data file.

Fig. 4. Structural comparison between SulE and its homologous proteins.

a Structure superposition of SulE, 4Q34, and esterase 713. The well superimposed α/β-hydrolase fold is colored gray. The varied parts are colored magenta for SulE, yellow for 4Q34, and green for esterase 713. b Superimposition of the catalytic site residues of 4Q34, esterase 713, and SulE. The residue positions are indicated in the order of 4Q34, esterase 713, and SulE, using the same color scheme as a. c Superposition of SulE (magenta) and esterase 713 (green). d Comparison between the substrate-binding pockets of SulE (magenta) and esterase 713 (green). MM and IBA are colored cyan and yellow, respectively.

Fig. 5. Structural basis for the altered activity of P44R mutant.

a, d, g The electron density of MM, CE, and TM was observed at the P44R/S209A/H33A active site. The 2Fo_Fc electron density map contoured at 1.0 σ level is shown as a blue mesh. b Superposition of the complex structure of S209A/H333A-MM (cyan) and P44R/S209A/H333A-MM (green). e Superposition of the complex structure of S209A/H333A-CE (magenta) and P44R/S209A/H333A-CE (white). h Superposition of the complex structure of S209A/H333A-TM (yellow) and P44R/S209A/H333A-TM (light blue). c, f, i Detailed analysis of the active site shown in panels b, e, h by using the same color scheme. Substrate molecules MM, CE, and TM are shown in the stick and sphere. Residues of interest in the active site are shown as sticks. Hydrogen bonds are shown in black dashed lines.

Fig. 6. SPR analysis of different sulfonylureas binding to wildtype (WT) and variant P44R.

a, b Binding of MM to WT SulE and variant P44R. The injected concentrations of MM were 0.156, 0.3125, 0.625, 1.25, 2.5, 5, and 10 μM, respectively. c, d Binding of CE to WT SulE and variant P44R. The injected concentrations of CE were 0.78, 1.56, 3.12, 6.25, and 12.5 μM, respectively. e, f Binding of TM to WT SulE and variant P44R. The injected concentrations of TM were 1.25, 2.5, 5, 10, and 20 μM, respectively. g, h Binding of BM to WT SulE and variant P44R. The injected concentrations of BM were 0.156, 1.25, 2.5, 5, 10, and 20 μM, respectively. SPR sensorgrams are provided as a Source Data file.

Fig. 7. Amino acid mutation analysis on lid loop.

a Effects of glycine and proline mutations in the lid loop on enzyme activity. b Effects of substitution of Pro44 by hydrophilic amino acids on enzyme activity. The relative enzyme activity of the variants to MM. Data are presented as mean values ± SD, n = 3. Error bars represent the standard deviation from three repeats. An unpaired two-tailed t-test was used to determine the statistical significance. *p = 0.0039 (G34A), *p = 0.016 (G49A), **p = 0.00013 (P33G), ***p = 4.8E − 06 (P38G), ***p = 1.7E − 05 (P41G), ***p = 6.3E − 05 (P44R), ***p = 1.3E − 05 (P44N), *p = 0.0020 (P44Q), **p = 0.00036 (P44K), ***p = 3.2E − 05 (P44S), and ***p = 2.0E − 05 (P44T). ns no significance. Source data are provided as a Source Data file.