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Identification of Six Novel Allosteric Effectors of Arabidopsis Thaliana Aspartate Kinase-Homoserine Dehydrogenase

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Identification of Six Novel Allosteric Effectors of Arabidopsis Thaliana Aspartate Kinase-Homoserine Dehydrogenase Identification of six novel allosteric effectors of Arabidopsis thaliana Aspartate kinase-Homoserine dehydrogenase. Physiological context sets the specificity Gilles Curien, Stéphane Ravanel, Mylène Robert, Renaud Dumas To cite this version: Gilles Curien, Stéphane Ravanel, Mylène Robert, Renaud Dumas. Identification of six novel allosteric effectors of Arabidopsis thaliana Aspartate kinase-Homoserine dehydrogenase. Physiological context sets the specificity. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2005, 280 (50), pp.41178-41183. 10.1074/jbc.M509324200. hal-00016689 HAL Id: hal-00016689 https://hal.archives-ouvertes.fr/hal-00016689 Submitted on 31 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 50, pp. 41178–41183, December 16, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Identification of Six Novel Allosteric Effectors of Arabidopsis thaliana Aspartate Kinase-Homoserine Dehydrogenase Isoforms PHYSIOLOGICAL CONTEXT SETS THE SPECIFICITY* Received for publication, August 24, 2005, and in revised form, October 3, 2005 Published, JBC Papers in Press, October 10, 2005, DOI 10.1074/jbc.M509324200 Gilles Curien1, Ste´phane Ravanel, Myle`ne Robert, and Renaud Dumas From the Laboratoire de Physiologie Cellulaire Ve´ge´tale UMR5168, CEA-CNRS-INRA-Universite´Joseph Fourier, 38054 Grenoble, France Downloaded from The Arabidopsis genome contains two genes predicted to code for genes (At1g31230 and At4g19710) for AK-HSDH enzymes. The cDNA bifunctional aspartate kinase-homoserine dehydrogenase enzymes from one of these genes (At1g31230) was previously cloned (4) but the (isoforms I and II). These two activities catalyze the first and the protein (AK-HSDH I) was not characterized. In a study from our group third steps toward the synthesis of the essential amino acids threo- (5), the cDNA corresponding to the second AK-HSDH gene www.jbc.org nine, isoleucine, and methionine. We first characterized the kinetic (At4g19710) was cloned and the protein (named AK-HSDH II) was and regulatory properties of the recombinant enzymes, showing overexpressed in E. coli, purified to homogeneity, and characterized. that they mainly differ with respect to the inhibition of the homo- To complete the analysis of Arabidopsis AK-HSDH enzymes the Ara- serine dehydrogenase activity by threonine. A systematic search for bidopsis thaliana AK-HSDH I isoform was cloned and overexpressed in at INRA Institut National de la Recherche Agronomique, on November 8, 2010 other allosteric effectors allowed us to identify an additional inhib- E. coli and its kinetic and regulatory properties were determined. In the itor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, course of this work, a systematic survey of the effect of other amino acids and valine) equally efficient on aspartate kinase I activity (4-fold led us to identify unexpected allosteric effectors of Arabidopsis Thr- activation). The six effectors of aspartate kinase I were all activators sensitive AKs. The key point of this discovery was the measurement of of aspartate kinase II activity (13-fold activation) and displayed a AK activities in the presence of low (i.e. physiological) concentrations of similar specificity for the enzyme. No synergy between different the substrates ATP and Asp. effectors could be observed. The activation, which resulted from a decrease in the K values for the substrates, was detected using low m MATERIALS AND METHODS substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other Construction of pET23AK-HSDH I Plasmid effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the The plasmid coding for AK-HSDH I devoid of its predicted transit presence of physiological concentrations of the seven allosteric peptide was constructed by PCR using the same strategy as for AK- effectors confirmed that aspartate kinase I and II activities were HSDH II (5). The initiating Met in recombinant AK-HSDH I was highly sensitive to changes in alanine and threonine concentrations. aligned with the position of the initiating Met in the recombinant AK- Thus, physiological context rather than enzyme structure sets the HSDH II (5). As part of the insertion of a NcoI restriction site, Gly62 and specificity of the allosteric control. Stimulation by alanine may play Ser63 were replaced by Met62 and Ala63 in the recombinant protein. the role of a feed forward activation of the aspartate-derived amino Sequencing of the cDNA was carried out and was in agreement with the acid pathway in plant. predicted cDNA sequence of At1g31230. Protein Expression and Purification The first and the third steps in the synthesis of the essential amino Transformed E. coli BL21pLys(S) cells were grown at 37 °C in 800 acids threonine (Thr) and methionine (Met) from aspartate (Asp) are ml of LB medium with 100 ␮g/ml carbenicillin and 34 ␮g/ml chlor- catalyzed in bacteria and in plants by the bifunctional enzyme aspartate 2 amphenicol. Isopropyl 1-thio-␤-D-galactopyranoside was added to a kinase-homoserine dehydrogenase (AK-HSDH) (E.C. 2.7.2.4-E.C. 1.1.1.3) (see Scheme 1). In Escherichia coli, two isoforms of AK-HSDH final concentration of 0.4 mM when bacterial growth was equivalent enzymes are found (thrA and metL gene products) (1). ThrA protein is to an A600 of 0.8. Cells were further grown for 15 h at 28 °C and inhibited by Thr on both activities. No allosteric effector could be iden- collected by centrifugation. The preparation of the crude extract and tified for the AK and the HSDH activities of the second isoform (MetL the first two purification steps (anion exchange chromatography and protein) (1). In plants, only one AK-HSDH isoform was identified after gel filtration) of AK-HSDH I were as for AK-HSDH II (5). AK-HSDH fractionation of crude extracts of soluble proteins (2, 3). However, the I eluted from the DEAE column at a higher KCl concentration (300 sequencing of the Arabidopsis genome revealed the existence of two mM) than AK-HSDH II (250 mM). After elution from the gel filtra- tion column, the protein was concentrated on a Centricon device (50 K) and loaded on a Green Sepharose column equilibrated with 25 mM * The costs of publication of this article were defrayed in part by the payment of page Hepes, pH 7.5, 10% (v/v) glycerol, and 1 mM Thr. AK-HSDH I protein charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. was excluded. Pure protein was quickly frozen in N2 and stored at 1 To whom correspondence should be addressed. Tel.: 33-4-38-78-23-64; Fax: 33-4-38- Ϫ80 °C for several months without any loss of activity. Recombinant 78-50-91; E-mail: [email protected]. 2 The abbreviations used are: AK, aspartate kinase; ASADH, aspartate semialdehyde AK-HSDH II protein was overexpressed in E. coli and purified as dehydrogenase; HSDH, homoserine dehydrogenase. previously reported (5). 41178 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280•NUMBER 50•DECEMBER 16, 2005 Arabidopsis Aspartate Kinase-Homoserine Dehydrogenase centration (10). Three extractions were performed for two independent purifications of chloroplasts to ensure reproducibility. RESULTS The cDNA corresponding to the At1g31230 gene from A. thaliana and predicted to code for a Thr-sensitive AK-HSDH was the first to be cloned (4), but the recombinant protein (named AK-HSDH I) was not characterized. Following the publication of the Arabidopsis genome, a second gene (At4g19710) predicted to code for a Thr-sensitive AK- HSDH was identified. In a previous study (5) from our group the corre- sponding recombinant protein (named AK-HSDH II) was overex- pressed in E. coli, purified to homogeneity, and characterized. In the present work, to determine the kinetic and regulatory properties of Downloaded from AK-HSDH I, the cDNA sequence corresponding to the predicted mature form of the enzyme (devoid of its predicted chloroplastic transit peptide) was amplified by PCR and cloned into a pET23d(ϩ) expression vector. The AK-HSDH I recombinant protein was expressed in E. coli as a soluble form but at a lower level than observed for AK-HSDH II (5). www.jbc.org Pure protein could be obtained after three purification steps (see “Mate- rials and Methods”). SCHEME 1. Aspartate pathway of amino acid biosynthesis. Each arrow represents an enzyme-catalyzed conversion. Dotted lines represent several steps. Comparison of AK-HSDH Isoform Kinetic Properties—AK I and AK II ϭ Ϯ Ϫ1 catalytic constants were similar (v/[E]0 5 0.5 s for AK I and v/[E]0 at INRA Institut National de la Recherche Agronomique, on November 8, 2010 Initial Velocity Measurements ϭ 6.2 Ϯ 0.5 sϪ1 for AK II). Compared with AK II, AK I displayed a higher All experiments were carried out in the spectrophotometer in a Km for aspartate and a lower Km for ATP, see TABLE ONE. quartz cuvette thermostated at 30 °C. HSDH activities were measured in the physiological direction (pro- AK Assay—AK activity was measured with a coupled assay using duction of homoserine from aspartate semialdehyde). The Km for aspar- aspartate semialdehyde dehydrogenase (ASADH) previously purified to tate semialdehyde proved to be very similar for both isoforms (290 Ϯ 17 homogeneity in the laboratory (6).
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