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

Xie, Yonghui; Wen, Xin; Zhao, Dongmei; Niu, Congwei; Zhao, Yuefang; Qi, Haoman; Xi, Zhen

doi:10.1002/cbic.201800367

Cited by 6 publications

(2 citation statements)

References 53 publications

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“…The CSs from plants and fungi already had certain enzyme activities that were comparable to that of Ec CSI. These relative activities for AHASs are consistent with our previous report and were determined by the A 525 method. We also chose a plant At CS to study in detail via the fluorogenic assay and found that the fluorescence changes were similar to that for Ec CSI (Figure S11), and the binding constants for probe 1 , ThDP, and FAD could be obtained from the fluorogenic assay as 87.0, 4.2, and 0.23 μM, respectively.…”

Section: Resultssupporting

confidence: 91%

Fluorogenic Assay for Acetohydroxyacid Synthase: Design and Applications

et al. 2019

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Acetohydroxyacid synthase (AHAS) exists in plants and many microorganisms (including gut flora) but not in mammals, making it an attractive drug target. Fluorescent-based methods should be practical for high-throughput screening of inhibitors. Herein, we describe the development of the first AHAS fluorogenic assay based on an intramolecular charge transfer (ICT)-based fluorescent probe. The assay is facile, sensitive, and continuous and can be applied toward various AHASs from different species, AHAS mutants, and crude cell lysates. The fluorogenic assay was successfully applied for (1) high-throughput screening of commerical herbicides toward different AHASs for choosing matching herbicides, (2) identification of a Soybean AHAS gene with broad-spectrum herbicide resistance, and (3) identification of selective inhibitors toward intestinal-bacterial AHASs. Among the AHAS inhibitors, an active agent was found for selective inhibition of obesity-associated Ruminococcus torques growth, implying the possibility of AHAS inhibitors for the ultimate goal toward antiobesity therapeutics. The fluorogenic assay opens the door for high-throughput programs in AHAS-related fields, and the design principle might be applied for development of fluorogenic assays of other synthases.

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Section: Resultssupporting

confidence: 91%

Fluorogenic Assay for Acetohydroxyacid Synthase: Design and Applications

et al. 2019

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“…Using solution NMR studies, our group had shown that IlvN interacts with the α and β domains of IlvB . These studies have been supported by recent biophysical and mutagenesis studies of the RSU–CSU interactions of E. coli AHASs . Thus, the CSU–RSU interaction is proposed to occur at the α–β interface on IlvB, with the residues on IlvN described above being the region of contact.…”

Section: Discussionmentioning

confidence: 99%

Crystallographic Structures of IlvN·Val/Ile Complexes: Conformational Selectivity for Feedback Inhibition of Aceto Hydroxy Acid Synthases

et al. 2019

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Conformational factors that predicate selectivity for valine or isoleucine binding to IlvN leading to the regulation of aceto hydroxy acid synthase I (AHAS I) of Escherichia coli have been determined for the first time from high-resolution (1.9-2.43 Å) crystal structures of IlvN•Val and IlvN•Ile complexes. The valine and isoleucine ligand binding pockets are located at the dimer interface. In the IlvN•Ile complex, among residues in the binding pocket, the side chain of Cys 43 is 2-fold disordered (χ 1 angles of gauche − and trans). Only one conformation can be observed for *

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An easy and sensitive assay for acetohydroxyacid synthases based on the simultaneous detection of substrates and products in a single step

Engelhardt,

Ebeling,

Kaltenegger

et al. 2024

Anal Bioanal Chem

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Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes the first step in the synthesis of the branched-chain amino acids valine, leucine, and isoleucine, pathways being present in plants and microorganisms, but not in animals. Thus, AHAS is an important target for numerous herbicides and, more recently, for the development of antimicrobial agents. The need to develop new and optimized herbicides and pharmaceuticals requires a detailed understanding of the biochemistry of AHAS. AHAS transfers an activated two-carbon moiety derived from pyruvate to either pyruvate or 2-oxobutyrate as acceptor substrates, forming 2-acetolactate or 2-acetohydroxy-2-butyrate, respectively. Various methods have been described in the literature to biochemically characterize AHAS with respect to substrate preferences, substrate specificity, or kinetic parameters. However, the simultaneous detection and quantification of substrates and unstable products of the AHAS-catalyzed reaction have always been a challenge. Using AHAS isoform II from Escherichia coli, we have developed a sensitive assay for AHAS-catalyzed reactions that uses derivatization with ethyl chloroformate to stabilize and volatilize all reactants in the aqueous solution and detect them by gas chromatography coupled to flame ionization detection or mass spectrometry. This assay allows us to characterize the product formation in reactions in single and dual substrate reactions and the substrate specificity of AHAS, and to reinterpret previous biochemical observations. This assay is not limited to the AHAS-catalyzed reactions, but should be applicable to studies of many metabolic pathways. Graphical Abstract

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

Cited by 6 publications

References 53 publications

Fluorogenic Assay for Acetohydroxyacid Synthase: Design and Applications

Fluorogenic Assay for Acetohydroxyacid Synthase: Design and Applications

Crystallographic Structures of IlvN·Val/Ile Complexes: Conformational Selectivity for Feedback Inhibition of Aceto Hydroxy Acid Synthases

An easy and sensitive assay for acetohydroxyacid synthases based on the simultaneous detection of substrates and products in a single step

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