Abstract
Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid (BCAA) biosynthesis, a pathway essential to the lifecycle of plants and microorganisms. This enzyme is of high interest because its inhibition is at the base of the exceptional potency of herbicides and potentially a target for the discovery of new antimicrobial drugs. The enzyme has conserved attributes from its predicted ancestor, pyruvate oxidase, such as a ubiquinone-binding site and the requirement for FAD as cofactor. Here, we show that these requirements are linked to the regulation of AHAS, in relationship to its anabolic function. Using various soluble quinone derivatives (e.g. ubiquinones), we reveal a new path of down-regulation of AHAS activity involving inhibition by oxidized redox-signaling molecules. The inhibition process relies on two factors specific to AHAS: (i) the requirement of a reduced FAD cofactor for the enzyme to be active and (ii) a characteristic slow rate of FAD reduction by the pyruvate oxidase side reaction of the enzyme. The mechanism of inhibition involves the oxidation of the FAD cofactor, leading to a time-dependent inhibition of AHAS correlated with the slow process of FAD re-reduction. The existence and conservation of such a complex mechanism suggests that the redox level of the environment regulates the BCAA biosynthesis pathway. This mode of regulation appears to be the foundation of the inhibitory activity of many of the commercial herbicides that target AHAS.
Highlights
Acetohydroxyacid synthase (AHAS)3 (E.C. 2.2.1.6) catalyzes the first step in de novo branched-chain amino acid (BCAA) biosynthesis, an anabolic pathway present in plants, fungi, and bacteria
We propose that the existence and conservation of such a complex mechanism is testimony to the regulation of the BCAA synthetic pathway by the redox level of the environment
AHAS activity is characterized by a temperature-dependent lag phase requiring incubation with substrate before reaching maximal activity [5]
Summary
AHAS activity is characterized by a temperature-dependent lag phase requiring incubation with substrate before reaching maximal activity [5]. The plateau of the curves corresponds to the catalytic rate constant (kact) of AHAS activation (POX activity), which has at 30 °C a value of 0.83 and 0.28 minϪ1 for ScAHAS and MtAHAS, respectively These results imply that at physiological concentrations of pyruvate, the rate of FAD reduction (and enzyme activation) is expected to be slow (e.g. if [pyruvate] Յ 1 mM, kobs(act) Յ 0.003/0.04 minϪ1 (corresponding to a half-time of activation Ն230/17 min) for MtAHAS/ScAHAS at 30 °C). The weak affinity of Q1 for MtAHAS implies that the inhibition relies mainly on two factors: (i) the rate at which Q1 oxidizes FAD and (ii) the rate of enzyme re-activation corresponding to the rate of FAD re-reduction by the POX side activity of AHAS Both factors are responsible for the observed time dependence of the inhibition (Fig. 6, A and B). An estimated value of 4.7 minϪ1 was obtained for kox at 30 °C (“Experimental Procedures”)
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