Structural basis of resistance to herbicides that target acetohydroxyacid synthase

Herbicides targeting acetohydroxyacid synthase (AHAS/ALS), the first enzyme of branched-chain amino acid biosynthesis, have been widely used for decades and span six chemical classes. Increasing field resistance has emerged at a limited number of residues in the AHAS active-site channel, with P197 and W574 being the most frequently observed. Prior structural and kinetic studies elucidated how various herbicide classes bind and inhibit AHAS, including a potent time-dependent accumulative inhibition mechanism driven by oxidative events at ThDP and FAD. However, no mutant–inhibitor complex structures existed, limiting understanding of resistance mechanisms at the molecular level. This study investigates how common resistance mutations (W574L, P197L, P197T, S653T) impact (i) herbicide binding affinity and (ii) time-dependent accumulative inhibition, combining detailed kinetics with crystallography to inform future herbicide design resilient to resistance.

Previous crystallographic work resolved AtAHAS complexes with 13 herbicides across five chemical classes, revealing shared binding features and the herbicide-induced oxidative inactivation mechanism (accumulative inhibition). The downregulation of AHAS by redox signaling (soluble quinones oxidizing FAD) and slow reactivation via the pyruvate oxidase side-reaction has been established. Computational and experimental discovery efforts (e.g., triazolopyrimidines, pyrimidine-biphenyl hybrids, MB-QSAR) expanded inhibitor space against resistant AHAS, but lacked structural data of herbicides bound to AHAS resistance mutants. Field data identify recurrent resistance at eight residues (notably P197 and W574) that contact inhibitors in the access channel/catalytic pocket. Together, these studies motivate direct structural analysis of mutant–herbicide complexes and comprehensive kinetic dissection of resistance.

Targets: AtAHAS mutants W574L, P197L, P197T, and S653T were generated by site-directed mutagenesis, expressed in E. coli BL21(DE3), and purified by IMAC and size-exclusion chromatography in ThDP/Mg2+/FAD-containing buffers. Purity was verified by SDS-PAGE.

Enzyme kinetics: Due to the AHAS lag phase, KM and kcat for pyruvate were determined using an implicit Michaelis–Menten formulation derived from substrate time courses (continuous spectrophotometric monitoring of pyruvate at 333 nm), avoiding initial-rate distortions at low pyruvate. Standard assay buffer contained 50 mM potassium phosphate pH 7.2, 1 mM ThDP, 10 mM MgCl2, 10 µM FAD; pyruvate 100–160 mM. Activation (kobs(act)) and redox regulation were assessed, including inhibition by ubiquinone Q0 (0.5 mM).

Inhibition assays: Competitive binding affinities (Ki) were measured for seven herbicides across four classes: sulfonylureas (chlorimuron-ethyl, amidosulfuron), triazolopyrimidines (metosulam, penoxsulam), pyrimidinylbenzoates (bispyribac, pyrithiobac), and imidazolinone (imazaquin). Time-dependent accumulative inhibition parameters were obtained by fitting progress curves to models yielding apparent inactivation (kiapp) and recovery (k3) rate constants.

Crystallography: Hanging-drop vapor diffusion crystallization used CHES/Na/K tartrate/(NH4)2SO4 conditions. X-ray data were collected at the Australian Synchrotron MX1, processed with XDS, and structures solved by molecular replacement (Phaser) and built in Coot; ligand dictionaries generated with PHENIX Elbow. Structures determined: uninhibited W574L (7U1U), W574L–bispyribac (7U25), W574L–chlorimuron-ethyl (7STQ), P197–CE (7UID), and P197–BS (7TZZ). Electron density showed oxidized/degraded ThDP species (ThThDP or ThAthDP) consistent with prior AHAS–herbicide complexes. Structural analyses compared mutant versus WT binding modes and assessed potential steric impacts on the ThDP oxidative chemistry space relevant to accumulative inhibition.

Catalysis and regulation: WT pyruvate KM = 12.5 ± 0.6 mM; mutants P197L/T and S653T were similar (12.4, 16.5, 15.5 mM), while W574L increased KM to 48.3 mM (~4-fold). WT kcat = 9.4 s−1; P197L 6.3 s−1, P197T 9.8 s−1, S653T 7.3 s−1; W574L increased to 25 s−1 (~2.5-fold). Redox regulation by Q0 was largely retained in mutants (slightly lower kobs(act), slightly greater inhibition by Q0), indicating mutants remain physiologically competent.

Binding affinities (Ki, nM): Mutations generally weakened binding, especially W574L which increased Ki 38-fold (imazaquin, IQ) up to ~35,000-fold (penoxsulam, PS). Examples (WT → W574L → P197L → P197T → S653T): CE 75 → 5200 → 4400 → 2200 → 53; AS 4200 → 870,000 → 93,500 → 61,400 → 492; PS 1.9 → 67,100 → 82 → 85 → 5; MT 29 → 39,300 → 1320 → 1130 → 66; BS 41 → 6100 → 115 → 106 → 90; PTB 179 → 91,200 → 2000 → 1500 → 218; IQ 18,500 → 704,000 → 13,800 → 12,400 → 240,000.

Accumulative inhibition: W574L abolished time-dependent accumulative inhibition for CE, AS, PS, and IQ, but not for BS. P197L/T abolished accumulative inhibition for CE, AS, PS; retained for BS and IQ. S653T increased accumulative inhibition efficiency (kiapp/k3) for AS (20→95), PS (87→126), and BS (18→39), but decreased for CE (226→69). Notably, BS induced virtually irreversible accumulative inhibition in W574L (k3 < 1×10−12 min−1; kiapp 0.03 min−1), akin to WT with IQ (k3 < 1×10−12 min−1; kiapp 0.86 min−1).

Structural mechanisms: W574L apo showed no global fold change aside from side-chain substitution. In W574L–CE, loss of π–π/halogen–π contacts with W574 reduces affinity (~70-fold) and rotates CE’s heterocycle (~38°), positioning Cl into the ThDP oxidative reaction space, creating steric clashes with modeled ThDP–peracetate/free peracetate (2.0–2.1 Å), explaining loss of accumulative inhibition. In W574L–BS, although π stacking with W574 is lost (affinity ~150-fold weaker), BS’s bulky, flexible, multi-ring scaffold maintains pose and allows ThAthDP/peracetate coexistence, preserving accumulative inhibition. P197T–CE retains most interactions but loses the proline–aromatic stabilization (estimated ΔΔG ~8.5 kJ/mol), reducing affinity and correlating with abolished accumulative inhibition. P197T–BS closely matches WT binding and directly contacts T197; Ki increases only ~3-fold and accumulative inhibition efficiency approximately doubles.

The findings reveal that herbicide resistance in AHAS is governed by two coupled mechanisms: decreased binding affinity and, critically, disruption of time-dependent accumulative inhibition that relies on oxidative chemistry at ThDP/FAD. Mutations at W574 and P197 reshape the local conformational landscape such that some herbicides encroach upon or obstruct the oxidative reaction space, abolishing accumulative inhibition despite retained binding. Bispyribac, with its bulky, flexible, multi-aromatic architecture, accommodates mutant-induced changes without occluding the oxidative space, sustaining or enhancing accumulative inhibition and explaining its robustness against site-of-action resistance. These mechanistic insights tie directly to field observations: W574L confers broad resistance across classes; P197L/T particularly compromise SUs and TPs but not IMIs; S653T mainly affects IMIs. The study underscores that preserving free space around ThDP for oxidative events is essential for durable AHAS inhibition and suggests design principles favoring flexible, multi-ring scaffolds that avoid the oxidative pocket.

This work provides the first mutant–herbicide structural views for common AHAS resistance mutations and integrates them with comprehensive kinetics to explain resistance. Key contributions include (i) quantifying large, mutation-specific losses in affinity, (ii) demonstrating that accumulative inhibition is frequently abolished by W574L and P197L/T but retained or enhanced by bispyribac, and (iii) identifying structural determinants (orientation and occupancy of the ThDP oxidative reaction space) that control accumulative inhibition. Design implications are clear: next-generation inhibitors should avoid intruding into the ThDP oxidative region while leveraging flexible, multi-aromatic motifs; natural product scaffolds like harzianic acid that exploit unused pocket regions are promising. Future research should expand mutant–inhibitor structural coverage across all classes, incorporate accumulative inhibition metrics into MB-QSAR and structure-based design, and evaluate combination formulations to delay resistance.

• Structural coverage was limited to selected mutant–herbicide pairs (e.g., W574L with CE/BS; P197 with CE/BS); not all mutants or herbicide classes were structurally characterized.
• Experiments employed the catalytic subunit; although prior holoenzyme data suggest conserved inhibitor binding sites, regulatory-subunit effects cannot be entirely excluded.
• In vitro kinetic parameters and accumulative inhibition behaviors may not fully capture in-field complexity; field correlation for PYBs is limited by a lack of reported mutant weed data.
• Only four of the six AHAS-inhibiting herbicide classes were tested here, potentially limiting generalizability across all classes.
• Some structures contained oxidized/degraded ThDP species (ThThDP/ThAthDP), which, while common in AHAS–herbicide crystallography, may differ from all catalytic states in solution.