Edited by F. Peter GuengerichAcetohydroxyacid 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.
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. AHAS catalyzes the condensation of pyruvate with another molecule of pyruvate, or with 2-ketobutyrate, to produce acetolactate or 2-acetohydroxybutyrate, respectively. In relationship to its essential role in anabolism, AHAS activity is highly regulated by cellular processes. For example, the activity of yeast AHAS (ScAHAS, catalytic subunit ilv2) is stimulated by a regulatory subunit (ilv6). The interaction between these two subunits confers sensitivity to feedback inhibition by valine, an inhibition that is partially reversed by ATP (1, 2) (Fig. 1).Of particular interest, soluble ubiquinones Q 0 (Q 0 ) and Q 1 (Q 1 ) inhibit enteric bacteria AHASs (ALS1-3) and this activity has been attributed to the presence of a quinone binding site derived from the evolution of AHAS from a pyruvate oxidase ancestor (3). The unexplained conservation of this "vestigial" quinone binding site in all AHAS suggests the possibility of its involvement in a, yet undiscovered, physiological role (3). Schloss (4) has also shown that Q 0 is able to oxidize the enzymebound FAD, a cofactor derived from a POX ancestor but without an assigned role in AHAS activity. Here, we demonstrate that (i) the reduction of the FAD cofactor is imperative to activate AHAS, and (ii) the inhibition of AHAS by uquinones is related to the oxidation of FAD. W...
