Structural basis of resistance to herbicides that target acetohydroxyacid synthase

Abstract

Acetohydroxyacid synthase (AHAS) is the target for more than 50 commercial herbicides; first applied to crops in the 1980s. Since then, 197 site-of-action resistance isolates have been identified in weeds, with mutations at P197 and W574 the most prevalent. Consequently, AHAS is at risk of not being a useful target for crop protection. To develop new herbicides, a functional understanding to explain the effect these mutations have on activity is required. Here, we show that these mutations can have two effects (i) to reduce binding affinity of the herbicides and (ii) to abolish time-dependent accumulative inhibition, critical to the exceptional effectiveness of this class of herbicide. In the two mutants, conformational changes occur resulting in a loss of accumulative inhibition by most herbicides. However, bispyribac, a bulky herbicide is able to counteract the detrimental effects of these mutations, explaining why no site-of-action resistance has yet been reported for this herbicide.

Crystal Structure of the Commercial Herbicide, Amidosulfuron, in Complex with Arabidopsis thaliana Acetohydroxyacid Synthase

“…13 Furthermore, the level of accumulative inhibition imposed by AS is better for the mutant than the wildtype enzyme; while the k 3 value (0.02 s −1 ) is identical for both enzymes, the k iapp is fivefold higher for the mutant compared to the wildtype enzyme (0.39 vs 1.9 min −1 ). 13 To help explain these observations, we determined the crystal structure of this mutant in complex with AS to 2.94 Å resolution (Table 1). As with the wildtype enzyme, a ThDP• peracetate adduct is observed in the active site (Figure 8), in agreement with the kinetic data that demonstrate the occurrence of accumulative inhibition.…”

“…Indeed, this is precisely what is observed when the crystal structure of the W574L mutant in complex with CE is superimposed on the crystal structure of the wildtype enzyme and, experimentally a complete loss of accumulative inhibition is also observed. 13 While GOLD was not successful in predicting this rotation, fitting using YASARA software showed some rotation of the R 2 group in that direction (Supporting Information, Figure 6). However, not enough to move into the space where peracetate would be located.…”

“…The results confirm and provide new experimental data to support the concept that accumulative inhibition is an important feature for herbicidal activity. 13 Based on this evidence, design strategies to discover new AHAS-inhibiting herbicides should consider avoiding the space where the oxygenase side-reaction occurs. Furthermore, we have shown that a member of the sulfonylurea family can be a weak inhibitor of AtAHAS (K i of 4.2 μM) but can also have potent herbicidal activity if it also possesses accumulative inhibition.…”

“…To date, 17 structures of wildtype or mutant Arabidopsis thaliana AHAS ( At AHAS) in complex with herbicides have been solved . Among these, seven are from the SU family (i.e., chlorimuron ethyl, metsulfuron methyl, chlorsulfuron, sulfometuron methyl, tribenuron methyl, monosulfuron, and monosulfuron-ester). , Their binding modes to At AHAS have many similarities, including the fact that they adopt an overall “L” shape, with the ends of the L containing the R 1 and R 2 groups of the SU scaffold (Figure ) and the central portion of the L forms the sulfonyl bridge.…”

“…The binding location of the SUs is strongly conserved such that the R 2 group forms π–π stacking interactions with W574, ,,− and the functional groups attached to this heterocycle point toward the two different cofactors, Mg 2+ ·ThDP and FAD. These herbicides also form contacts with the side chains of residues that include A122, V196, P197, M200, A205, D376, K256, R377, and S653. , …”