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

Abstract

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...

High Resolution Crystal Structures of the Acetohydroxyacid Synthase‐Pyruvate Complex Provide New Insights into Its Catalytic Mechanism

“…However, a time‐course experiment shows that the CW EPR signal increases in intensity during the lag‐phase (Figure C). Because the rate of the POX side reaction decreases during the lag‐phase, in correlation with the decrease of the rate of FAD reduction, it is evident that the EPR signal observed here does not arise form the POX activity but rather from the conversion of pyruvate to 2‐acetolactate for which the rate of this reaction increases during the lag‐phase. With the current data we cannot unambiguously assign the EPR signal to a FAD radical or to a superoxide radical .…”

“…The answer most likely resides in requirement of FAD‐dependent AHAS to be regulated by the redox level of the environment. The mechanism of regulation involves the oxidation of the cofactor FAD red by reduced soluble quinones derivatives, a process that inactivates the enzyme . The catalytic mechanism proposed here perfectly explains why the oxidation of FAD red to FAD ox leads to the inactivation of AHAS.…”

“…These indications suggest the involvement of a redox reaction during catalysis. As the oxidation of FAD red to FAD ox results in the inactivation of AHAS,, the only feasible conclusion is a one‐electron redox reaction involving FAD red /FAD radical as a redox couple. This scenario is strongly supported by EPR showing that the production of a radical occurs when Sc AHAS reacts with pyruvate (Figure ), a situation that could not happen in a mechanism only involving ThDP (Figure ).…”

“…The existence of a tunnel that allows O 2 molecules to access the active site independently of substrate avoiding the proximity of FAD is an evolutionary feature that supports the present mechanism. Indeed, it is crucial that FAD red avoids the contact with free molecules of O 2 as it would interfere with the one‐electron redox reaction involving FAD, leading to the oxidation of FAD red to FAD ox with concomitant inactivation of the enzyme …”

“…Tittmann, Schroder, Golbik, McCourt, Kaplun, Duggleby, Barak, Chipman, Hubner, have shown that in a side reaction of AHAS, HE‐ThDP can transfer two electrons to the adjacent FAD in an intramolecular redox reaction yielding 2‐acetyl‐ThDP and FAD red , concluding that AHAS and POX share a common POX‐like ancestor that catalyses a reaction that relies on electron transfer between ThDP and FAD. Lonhienne, Garcia, and Guddat, have shown that the reduction of FAD by the POX side reaction is imperative to activate the enzyme (Figure ), as FAD ox cannot sustain the AHAS reaction. Moreover, a new high resolution structure of the free yeast AHAS ( Sc AHAS) revealed that the isoalloxazine ring of FAD takes different conformations in the active sites of the dimer, one being bent by 21° across the N5‐N10 axis and expected to represent reduced FAD [FAD red , either FADH 2 or FADH − )], and the other being flat, representing oxidized FAD (FAD ox ) or semi‐reduced FAD (FAD radical , either FAD − or FADH . )…”