Allosteric threonine synthase: reorganization of the PLP site upon asymmetric activation through SAM binding to a novel site Corine Mas-Droux, Valérie Biou, Renaud Dumas To cite this version: Corine Mas-Droux, Valérie Biou, Renaud Dumas. Allosteric threonine synthase: reorganization of the PLP site upon asymmetric activation through SAM binding to a novel site. Journal of Biologi- cal Chemistry, American Society for Biochemistry and Molecular Biology, 2006, 281, pp.5188 -5196. 10.1074/jbc.M509798200. hal-00016704 HAL Id: hal-00016704 https://hal.archives-ouvertes.fr/hal-00016704 Submitted on 30 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. Copyright THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 8, pp. 5188–5196, February 24, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Allosteric Threonine Synthase REORGANIZATION OF THE PYRIDOXAL PHOSPHATE SITE UPON ASYMMETRIC ACTIVATION THROUGH S-ADENOSYLMETHIONINE BINDING TO A NOVEL SITE*□S Received for publication, September 7, 2005, and in revised form, November 14, 2005 Published, JBC Papers in Press, November 29, 2005, DOI 10.1074/jbc.M509798200 Corine Mas-Droux‡, Vale´rie Biou§1, and Renaud Dumas‡2 From the ‡Laboratoire de Physiologie Cellulaire Ve´ge´tale, De´partement Re´ponse et Dynamique Cellulaires, CNRS Commissariat a` l’Energie Atomique, 38054 Grenoble, France and §Laboratoire d’Enzymologie et de Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France Threonine synthase (TS) is a fold-type II pyridoxal phosphate an allosteric activation by S-adenosylmethionine (AdoMet), the end Downloaded from (PLP)-dependent enzyme that catalyzes the ultimate step of threo- product of the methionine pathway. Indeed, previous biochemical nine synthesis in plants and microorganisms. Unlike the enzyme results showed that allosteric binding of AdoMet leads to an 8-fold from microorganisms, plant TS is activated by S-adenosylmethi- increase in the rate of catalysis and to a 25-fold decrease in the Km value onine (AdoMet). The mechanism of activation has remained for OPH in the Arabidopsis thaliana enzyme (2). This activation is unknown up to now. We report here the crystallographic structures correlated with conformational modifications of the PLP environment http://www.jbc.org/ of Arabidopsis thaliana TS in complex with PLP (aTS) and with PLP as demonstrated by fluorescence experiments (2). and AdoMet (aTS-AdoMet), which show with atomic detail how The structure of the apoform of A. thaliana TS (called apo-aTS here- AdoMet activates TS. The aTS structure reveals a PLP orientation after) has been solved previously at 2.25 Å resolution (3). A. thaliana never previously observed for a type II PLP-dependent enzyme and threonine synthase is a dimer with 486 amino acids/monomer. The explains the low activity of plant TS in the absence of its allosteric structure of the apoenzyme shows a symmetric, compact dimer with at INRA Institut National de la Recherche Agronomique on May 31, 2019 activator. The aTS-AdoMet structure shows that activation of the four domains in each monomer: domain 1 (residues 1–130), domain 2 enzyme upon AdoMet binding triggers a large reorganization of (residues 161–270), domain 3 (residues 131–160 and 271–460), and the active site loops in one monomer of the structural dimer and allows C-terminal swap domain, thus called because it forms a structural the displacement of PLP to its active conformation. Comparison domain with domain 2 of the other monomer (residues 461–486). The with other TS structures shows that activation of the second mon- active site is located in a deep crevice at the interface of domains 2 and omer may be triggered by substrate binding. This structure also 3 of each monomer. discloses a novel fold for two AdoMet binding sites located at the PLP-dependent enzymes catalyze a large variety of reactions, dimer interface, each site containing two AdoMet effectors bound mostly on amino acid substrates. TS belongs to the family of fold- in tandem. Moreover, aTS-AdoMet is the first structure of an type II PLP-dependent enzymes (4). Comparison of the plant enzyme enzyme that uses AdoMet as an allosteric effector. structure with the structures of the non-allosteric TS from Thermus thermophilus (5), Saccharomyces cerevisiae (6), or with the struc- Threonine synthase (TS)3 is involved in the essential amino acids tures of other fold-type II enzymes (for example, threonine deami- pathway derived from aspartate and catalyzes the conversion of nase (7), O-acetylserine sulfhydrylase (8), or tryptophan synthase O-phosphohomoserine (OPH) into threonine and inorganic phosphate (9)) showed that all of these proteins share a common fold for via a pyridoxal phosphate (PLP)-dependent reaction. Plant TS is a fas- domains 2 and 3, whereas domain 1 and the swap domain are specific cinating enzyme. Unlike bacteria and fungi, its substrate, OPH, is a to TS. However, the structure of the plant apo-aTS revealed an branch point intermediate between the methionine and threonine path- absence of electron density for PLP in the active site. In addition, ways. Because of its specific role in the partitioning of methionine and several amino acids that interact with the PLP in enzymes belonging threonine (1), plant TS, which has a low basal activity rate, has acquired to the fold-type II family exhibit different orientations in the apo- aTS structure. Furthermore, a large loop that tops the active site and * The costs of publication of this article were defrayed in part by the payment of page is ordered in all structures of fold-type II enzymes is disordered in charges. This article must therefore be hereby marked “advertisement” in accordance the plant TS structure between residues 341 and 362. From these with 18 U.S.C. Section 1734 solely to indicate this fact. □S The on-line version of this article (available at http://www.jbc.org) contains supple- observations, the following three hypotheses were made. First, the mental Figs. 1–4 and references. apo-aTS structure previously determined might correspond to an The atomic coordinates and structure factors (codes 2C2B and 2C2G) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers Uni- inactive form that can only bind its PLP in a conformation unfavor- versity, New Brunswick, NJ (http://www.rcsb.org/). able for catalysis. Second, the activator AdoMet should lead to a 1 To whom correspondence may be addressed: Laboratoire d’Enzymologie et de Bio- change in the PLP orientation to one similar to that observed in the chimie Structurales CNRS, avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France. Tel.: 33-0-1-69-82-34-81; Fax: 33-0-1-69-82-31-29; E-mail: [email protected]. fold-type II enzymes. Third, the above conformational modifications 2 To whom correspondence may be addressed: Laboratoire de Physiologie Cellulaire should promote the enzyme activation. Ve´ge´tale, De´partement Re´ponse et Dynamique Cellulaires. Commissariat a`l’Energie Atomique, 38054 Grenoble, France. Tel.: 33-0-4-38-78-23-58; Fax: 33-0-4-38-78-50-91; To check these hypotheses, we have determined the crystallographic E-mail: [email protected]. structures of the A. thaliana TS in the presence of PLP and with PLP and 3 The abbreviations used are: TS, threonine synthase; AP5, 2-amino-5-phosphonopen- tanoic acid; OPH, O-phosphohomoserine; PLP, pyridoxal phosphate; AdoMet, S-ad- AdoMet. Along with the structure of holoenzyme TS-PLP (called aTS enosylmethionine; aTS, A. thaliana threonine synthase complexed with PLP; aTS- hereafter), we present here the first structure of a complex between an AdoMet, A. thaliana threonine synthase complexed with PLP and AdoMet; tTS, T. thermophilus threonine synthase; r.m.s., root mean square; MES, 4-morpho- enzyme and AdoMet as an allosteric activator (hereafter called lineethanesulfonic acid. aTS-AdoMet). 5188 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 8•FEBRUARY 24, 2006 Novel AdoMet and PLP Binding Sites in Threonine Synthase EXPERIMENTAL PROCEDURES TABLE 1 Purification and Crystallization—A. thaliana aTS was purified as Data collection and refinement described previously (10). The purified protein was concentrated to 18 The numbers in parentheses indicate the value in the outer resolution shell. The Rfree was calculated using 5% of reflections, which were kept apart from the refine- mg/mlin20mM HEPES-KOH, pH 7.5. ment during the whole process. R.m.s., root mean square. To collect the aTS data set, TS was crystallized as described in Ref. 3, aTS aTS-SAM with reservoirs containing 1 M LiCl, 13% (w/v) polyethylene glycol 6000, Diffraction data 5mM dithiothreitol, 0.1 M MES-KOH buffer at pH 6.5 and 6 mM PLP. 2 Wavelength (Å) 1.54 0.934 ␮ ␮ Resolution (Å) 2.6 2.6 l of protein at 10 mg/ml were mixed with 2 l of reservoir solution. Space group P1 C2 The crystals grew for 3 weeks before they reached a size of ϳ500 ␮m. No. of reflections 51,025 (7,279) 891,931 (52,594) No. of unique reflections 25,774 (3,698) 98,317 (14,300) They were transferred into a solution containing 1 M LiCl, 20% (w/v) Completeness 95.5 (95.5) 99.8 (99.8) polyethylene glycol 6000, 5 mM dithiothreitol, 0.1 M MES-KOH, 10 Multiplicity 2 (2) 3.7 (3.7) a mg/ml protein, and 6 mM PLP and incubated for 3 h before being trans- Rsym (%) 5.4 (29.0) 8.3 (44.4) ferred to a cryobuffer containing 10% glycerol, 1 M LiCl, 20% (w/v) Structure refinement Resolution limits (Å) 20–2.6 30–2.6 Downloaded from polyethylene glycol 6000, 5 mM dithiothreitol, 6 mM PLP, and 0.1 M b Rfactor (%) 18.5 20.6 MES-KOH, pH 6.5.