Construction of an active acetohydroxyacid synthase I with a flexible linker connecting the catalytic and the regulatory subunits

Vyazmensky, Maria; Engel, Stanislav; Kryukov, Olga; Berkovich-Berger, Dvora; Kaplun, Ludmila

doi:10.1016/j.bbapap.2006.02.011

Cited by 10 publications

(11 citation statements)

References 16 publications

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“…Mutations were introduced into plasmid pQE‐BN [34] by the method of PCR overlap extension [35]. Each plasmid used for protein expression was sequenced.…”

Section: Methodsmentioning

confidence: 99%

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate–thiamin diphosphate adduct in AHAS I

et al. 2012

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Acetohydroxy acid synthase (AHAS; http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/2/1/6.html) is a thiamin diphosphate (ThDP)‐dependent decarboxylase‐ligase that catalyzes the first common step in the biosynthesis of branched‐chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl‐ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2‐ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2‐ketobutyrate (2‐KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing–quench NMR method or stopped‐flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2‐lactyl‐ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2‐KB, putting into question whether AHAS operates via a pure ping–pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor–acceptor:enzyme complex followed by covalent, ThDP‐promoted catalysis with concerted decarboxylation–carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS.

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“…Mutations were introduced into plasmid pQE‐BN [34] by the method of PCR overlap extension [35]. Each plasmid used for protein expression was sequenced.…”

Section: Methodsmentioning

confidence: 99%

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate–thiamin diphosphate adduct in AHAS I

et al. 2012

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“…Thrombin protease was obtained from Novagen (USA). The pQEBNwt plasmid (containing the E. coli AHASⅠcatalytic and regulatory subunit gene) [26] , pUIwt and pET-NHisHwt plasmid (containing the E. coli AHAS Ⅲ catalytic and regulatory subunit gene, respectively) [20,21] were kindly provided by Professor David M. Chipman (Ben-Gurion University of the Negev). Abbreviation used in the paper for different subunits are as follows: 1C denotes the catalytic subunits of AHASⅠ ; 1R denotes the regulatory subunit of AHASⅠ; 1CR denotes the AHASⅠholoenzyme contained CSU and RSU which were expressed from a single operon; 3C denotes the catalytic subunit of AHAS Ⅲ; 3R denotes the regulatory subunit of AHAS Ⅲ; 3CR denotes the AHAS Ⅲ holoenzyme; CHis6 and NHis6 suggest His-tag at C-terminal and N-terminal of protein, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The genes coding for AHASⅠas 1CR, 1C, and 1R were subcloned from pQE-BNwt plasmid [26] . The genes coding for AHAS Ⅲ as 3C and 3R were subcloned from pUIwt and pET-NHis-Hwt plasmid.…”

Section: Cloning Of the Expression Plasmidsmentioning

confidence: 99%

“…The multidimensional multinuclear NMR investigations indicated that E. coli ilvN (the regulatory subunit of AHASⅠ) interacted with the FAD binding domain of ilvB (the catalytic subunit of AHASⅠ) and activated the AHAS Ⅰ enzyme [23] . Although crystal structures of catalytic subunits of yeast [24] and A. thaliana [25] and regulatory subunit of E. coli AHAS Ⅲ [22] have been known, the structural basis for the interaction of the regulatory subunit with the catalytic subunit in the case of E. coli AHAS was less known, except that an active AHASⅠwith a flexible linker connecting the catalytic and the regulatory subunits was created by Vyazmensky et al [26] . Quantitative analysis of the interaction between catalytic and regulatory subunits was scarce, which prompted us to examine the processes between catalytic and regulatory subunits of E. coli AHAS on the basis of the surface plasmon resonance (SPR) technique.…”

Section: Introductionmentioning

confidence: 99%

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Homologous and heterologous interactions between catalytic and regulatory subunits of Escherichia coli acetohydroxyacid synthase I and III

Niu

Feng

Zhou

et al. 2009

Sci. China Ser. B-Chem.

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Homologous and heterologous interactions between acetohydroxyacid synthase (AHAS) I and Ⅲfrom E. coli have been studied by surface plasmon resonance (SPR). The catalytic and regulatory subunits association for AHAS I (K D = 1.13 × 10 −7 M) was stronger than that for AHAS Ⅲ (K D = 5.29 × 10 −7 M). A strong heterologous association between regulatory and catalytic subunits and heterologous activation of catalytic subunits were observed. SPR results combined with enzyme kinetics indicate that the reconstituted heterologous enzymes had similar kinetic properties as homologous enzymes, implying that the regulatory subunit of AHAS I could be replaced by the regulatory subunit of AHAS Ⅲ and vice versa. This work may be useful to further understandings of the mechanism of regulation of AHAS.acetohydroxyacid synthase, subunit interaction, surface plasmon resonance

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“…The structural basis for the interaction between RSU and CSU is also less well understood 15. Vyazmensky et al found that an AHAS I created by connecting CSU and RSU with a flexible linker was active,16 but the crystallization of this single‐chain AHAS I has not been reported. Through study of each isolated domain of ilvB (CSU of E. coli AHAS I) by multidimensional solution NMR, the Sarma group concluded that ilvN (RSU of E. coli AHAS I) interacts with the FAD binding domain of ilvB 6.…”

Section: Introductionmentioning

confidence: 99%

Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

et al. 2012

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Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein-protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins.

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Construction of an active acetohydroxyacid synthase I with a flexible linker connecting the catalytic and the regulatory subunits

Cited by 10 publications

References 16 publications

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate–thiamin diphosphate adduct in AHAS I

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate–thiamin diphosphate adduct in AHAS I

Homologous and heterologous interactions between catalytic and regulatory subunits of Escherichia coli acetohydroxyacid synthase I and III

Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

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