Structural insights into the mechanism of inhibition of AHAS by herbicides

Abstract

Acetohydroxyacid synthase (AHAS), the first enzyme in the branched amino acid biosynthesis pathway, is present only in plants and microorganisms, and it is the target of >50 commercial herbicides. Penoxsulam (PS), which is a highly effective broad-spectrum AHAS-inhibiting herbicide, is used extensively to control weed growth in rice crops. However, the molecular basis for its inhibition of AHAS is poorly understood. This is despite the availability of structural data for all other classes of AHAS-inhibiting herbicides. Here, crystallographic data for AHAS (2.3 Å) and AHAS (2.5 Å) in complex with PS reveal the extraordinary molecular mechanisms that underpin its inhibitory activity. The structures show that inhibition of AHAS by PS triggers expulsion of two molecules of oxygen bound in the active site, releasing them as substrates for an oxygenase side reaction of the enzyme. The structures also show that PS either stabilizes the thiamin diphosphate (ThDP)-peracetate adduct, a product of this oxygenase reaction, or traps within the active site an intact molecule of peracetate in the presence of a degraded form of ThDP: thiamine aminoethenethiol diphosphate. Kinetic analysis shows that PS inhibits AHAS by a combination of events involving FAD oxidation and chemical alteration of ThDP. With the emergence of increasing levels of resistance toward front-line herbicides and the need to optimize the use of arable land, these data suggest strategies for next generation herbicide design.

Commercial AHAS-inhibiting herbicides are promising drug leads for the treatment of human fungal pathogenic infections

“…S6). Herbicide (e.g., PB, SCT, and TP families)-induced modifications to ThDP have also been observed for AtAHAS (23,24), and these are the result of oxidative reactions that promote the formation of peracetate in the active site (24) and the subsequent alteration of ThDP (Fig. 3).…”

“…Our kinetic, structural, and MS data suggest that, upon herbicide binding, ThDP is primarily altered into two analogs: ThThDP or ThAthDP. Turnover conditions and molecular oxygen [which binds AHAS nearby ThDP (59)] are required for this to occur (23,24), implying that the presence of the hydroxyethyl (He) ThDP intermediate in the active site is required for either herbicide binding and/or the generation of peracetate, a product of the oxygenase activity of AHAS (60). From these results, the following mechanism has been developed.…”

“…Our kinetic assays concluded that CE and several other AHAS inhibitors of the SU and TP families show potent inhibition of CaAHAS and CnAHAS that also correlates with strong antifungal activity. The ability of the herbicides to induce accumulative inhibition via ThDP alteration and FAD oxidation (23,24) is an important determinant in how effective these compounds are in biological assays, so should be a key factor in consideration of the design of AHAS inhibitors to act as antifungal drugs. Importantly, CE stands out over all commercial herbicides tested in this study because it possesses a broad spectrum of antifungal activity against five different Candida species and C. neoformans and is fungicidal against C. albicans in vitro.…”

Interactions between the ACT Domains and Catalytic Subunits of Acetohydroxyacid Synthases (AHASs) from Different Species

“…Sequences of the CSs are largely conserved (≈40 % similarity) in AHASs from different species. The X‐ray crystal structures of CSs from Saccharomyces cerevisiae ( Sc CS) or Arabidopsis thaliana ( At CS) show that a CS monomer contains three distinct domains (α, β, and γ domains). The active site of the CS is located at the interface between two monomers, which implies that the minimum quaternary structure for catalysis should be a dimer.…”

Engineering Herbicide-Tolerance Rice Expressing an Acetohydroxyacid Synthase with a Single Amino Acid Deletion

“…Herbicide tolerance was generated when some mutations weakened the binding between AHAS and an herbicide [14]. A SU/PYB herbicide was bound to the W548 with a π-π interaction [27,28]. When the W548 was replaced with a non-aromatic residue, the π-π interaction disappeared and herbicide tolerance was generated [29,30].…”