Commercial Herbicides Can Trigger the Oxidative Inactivation of Acetohydroxyacid Synthase

Abstract

Acetohydroxyacid synthase (AHAS) inhibitors are highly successful commercial herbicides.N ew kinetic data show that the binding of these compounds leads to reversible accumulative inhibition of AHAS.C rystallographic data (to aresolution of 2.17 ) for an AHAS-herbicide complex shows that closure of the active site occurs when the herbicidal inhibitor binds,t hus preventing exchange with solvent. This feature combined with new kinetic data shows that molecular oxygen promotes an accumulative inhibition leading to the conclusion that the exceptional potency of these herbicides is augmented by subversion of an inherent oxygenase side reaction. The reactive oxygen species produced by this reaction are trapped in the active site,triggering oxidation reactions that ultimately lead to the alteration of the redox state of the cofactor flavin adenine dinucleotide (FAD), af eature that accounts for the observed reversible accumulative inhibition.Acetohydroxyacid synthase (AHAS;E C2 .2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway,catalyzes either the conversion of two molecules of pyruvate into 2-acetolactate,orone molecule of pyruvate and one molecule of 2-ketobutyrate to 2-aceto-2-hydroxybutyrate ( Figure 1). This enzyme is found in plants,bacteria, and fungi but not in animals,m aking it an attractive target for biocide discovery.I ndeed, inhibitors of AHAS have been highly successful commercial herbicides for more than thirty years. [1] However,alack of clarity surrounds the mechanism of inhibition of AHAS by these herbicides.T he reaction has been reported to be time-dependent and biphasic, [2] corresponding to slow-binding inhibition. However,inAHAS,this process has only been observed under turnover conditions, [3] suggesting that there may be an alternative explanation for the observed time-dependent and biphasic inhibition profiles. Adding to the conjecture,t he inhibition of AHAS by herbicides has,i ns ome cases,b een reported to be irreversible, [2a,4] which is inconsistent with am echanism of slowbinding inhibition. In addition, it is also feasible that thiamin diphosphate (ThDP;aBreslow intermediate), ac ofactor in the active site,can participate in alternative reactions with the herbicide (for example by protonation of the enamine or by formation of the keto form through tautomerization), [5] thereby influencing inhibition.Herein, we report our investigations into the process of AHAS inhibition at suboptimal concentrations using penoxsulam (PS;F igure 1B), as al ead example of the triazolopyrimidine sulfonamide family of herbicides.T he kinetic curves (Figure 2A)resemble those reported previously for inhibitors of AHAS where the time dependence of the inhibition was accredited to reversible slow-binding inhibition. [2c,6] However, in this case,s low-binding inhibition is not involved because the amount of inhibition of AHAS at low concentrations of inhibitor exceeds stoichiometric equivalence.F or example,at ac oncentration of 25 nm,P Si nactivated approximately 1 mm ...

The Role of a FAD Cofactor in the Regulation of Acetohydroxyacid Synthase by Redox Signaling Molecules

“…In the first phase, the reduction is rapid and involves around 25-30% of the total FAD. This phase could be attributed to the pre-existence of an enzyme-hydroxyethylThDP complex allowing rapid reduction of FAD ox through a long-range electron transfer (7). In the second phase, the FAD reduction rate is lower and follows an exponential decay.…”

“…Indeed, the quinone reduction activity would allow AHAS to quickly re-establish normal redox conditions in the cell, restoring normal AHAS activity. Besides these differences, there is a commonality between quinones and the commercial herbicides in that their mode of inhibition is time-dependent and linked to oxidation of the FAD red (7). However, the mechanisms leading to this common phenomenon are unrelated.…”

Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families

“…Previously, we have shown that partial O 2 depletion decreases the amount of accumulative inhibition (9), and that condition was used here for measurement of K i values. For illustration, the inhibition of AtAHAS by PB under partial anaerobic conditions (Fig.…”

“…Based on these data, several general conclusions could be made: (i) the k iapp and k 3 rates are variable and there is no obvious correlation between k iapp and k 3 values, in agreement with these two rates representing independent events. k iapp represents the apparent rate of inactivation in presence of the inhibitor, whereas k 3 represents the enzyme recovery rate that occurs in absence of inhibitor (9). (ii) The k 3 value of IQ is so small that it is effectively zero.…”

“…It has been ascertained that inhibition of fungal AHAS by these herbicides fits a model of reversible accumulative inhibition (9). Therefore, two general aspects need to be considered when studying inhibition of AHAS: (i) the affinity of the inhibitor for the enzyme (K i ) and (ii) the effect of accumulative inhibition.…”