Báo cáo khoa học: Allosteric monofunctional aspartate kinases from Arabidopsis pptx

Allosteric monofunctional aspartate kinases from Arabidopsis Gilles Curien, Mathieu Laurencin, Myle ` ne Robert-Genthon and Renaud Dumas Laboratoire de Physiologie cellulaire Ve ´ ge ´ tale (PCV-DRDC), CEA-CNRS-INRA-Universite ´ Joseph Fourier, Grenoble, France The essential amino acids Lys, Met and Thr and the methylating agent S-adenosyl-l-methionine (SAM) are derived in plant and bacteria from Asp. The first step of this branched metabolic pathway consists of the activation of Asp to aspartyl phosphate in the presence of ATP. This reaction is catalyzed by aspartate kinase (AK; EC 2.7.2.4). The number of AK isoforms varies greatly among different organisms (from one in yeast, to at least five in plants). A fascinating aspect of AK is the existence of very different allosteric control patterns, depending on the source organisms and the isoforms considered. Plant monofunctional AK activity is inhibited by Lys, as reported for bacterial AKs, but displays an additional feature that is specific to the plant enzyme. Indeed, activity measurements carried out on protein extracts from various plants revealed that Lys-sensitive AK activity is inhibited in a synergistic manner by Keywords aspartate kinase; lysine; S-adenosyl- L-methionine; slow inhibition, synergy Correspondence G. Curien, Laboratoire de Physiologie cellulaire Ve ´ ge ´ tale (PCV-DRDC), 17 avenue des Martyrs, 38054 Grenoble, France Fax ⁄ Tel: +33 4 38 78 50 91 E-mail: gcurien@cea.fr (Received 21 September 2006, revised 31 October 2006, accepted 6 November 2006) doi:10.1111/j.1742-4658.2006.05573.x Plant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, aspartate kinase 1 displayed low apparent affinity for lysine compared to aspartate kinase 2 and aspartate kinase 3. In the presence of SAM, the apparent affin- ity of aspartate kinase 1 for lysine increased considerably, with K 0.5 values for lysine inhibition similar to those of aspartate kinase 2 and aspartate kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent K m values for the substrates ATP and aspartate. The mechan- ism of aspartate kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accord- ance with equilibrium binding analyses indicating that SAM binding to aspartate kinase 1 requires prior binding of lysine. The three-dimensional structure of the aspartate kinase 1–Lys–SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell 18, 1681–1692]. Taken together, the data suggest that, upon binding to the inactive aspartate kinase 1–Lys complex, SAM promotes a slow conformational transition leading to formation of a stable aspartate kinase 1–Lys–SAM complex. The increase in aspartate kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between aspartate kinase 1 and aspartate kinase 1–Lys towards the inactive form. Abbreviations AK, aspartate kinase; SAM, S-adenosyl- L-methionine. 164 FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS SAM and Lys [1,2]. The mechanism of plant AK synergistic inhibition by Lys and SAM has never been characterized, and the exact function of this plant-spe- cific control is not clear. The publication of the Ara- bidopsis genome complicated the matter further. Three genes potentially coding for monofunctional AK enzymes exist in this plant (At5g13280 for AK1; At5g14060 for AK2; At3g02020 for AK3). The corres- ponding proteins have never been characterized at a biochemical level. It is thus still unclear whether mono- functional AKs are all synergistically inhibited by Lys and SAM. In addition, we recently demonstrated that plant bifunctional AK–homoserine dehydrogenases from Arabidopsis thaliana (isoforms I and II) are acti- vated by various amino acids [3]. Whether monofunc- tional AKs from plants are sensitive to these activators is unknown. In order to answer these questions and to characterize the mechanism of the plant-specific syner- gistic inhibition of AK by Lys plus SAM, the three Arabidopsis cDNAs potentially coding for monofunc- tional AK enzymes were cloned, and the correspond- ing proteins were overproduced in Escherichia coli, purified to homogeneity and characterized. This work allowed us to show that only one AK (AK1) is inhib- ited in a synergistic manner by Lys and SAM, and to characterize in detail the nature of the inhibition of the plant AKs by Lys. AK1 was cocrystallized in the pres- ence of Lys and SAM, and the structure of the com- plex was solved in this laboratory [4], representing the first structure of an AK. The present kinetic analyses complement this structural analysis. Results cDNA cloning, overproduction of the recombinant enzymes in E. coli and purification The predicted amino acid sequences of AK1, AK2 and AK3 contain putative N-terminal plastid-targeting sequences, in accordance with the chloroplast localiza- tion of AK activity [5]. In order to obtain recombinant enzymes in sufficient quantities for biochemical analy- ses, cDNAs encoding the mature enzymes (with the putative transit peptides removed) were cloned into bacterial overexpression plasmids. The sequences of the PCR-cloned cDNAs matched the published sequence [6]. All proteins were expressed in soluble forms in E. coli BL21 codon (+). On the first anion exchange column, AK3 was eluted at a much lower ionic strength (25 mS) than AK2 (40 mS) or AK1 (50 mS). A second purification step consisted of fractionation on a Q-Sepharose column for AK1 and gel filtration for AK2 and AK3. Ten to fifty micrograms of highly puri- fied proteins (Fig. 1) were obtained per liter of culture. On the denaturing gel documented in Fig. 1, the pro- teins migrated in agreement with their predicted molecular masses (AK1, 55.9 kDa; AK2, 53.2 kDa; and AK3, 55.1 kDa). All proteins were stable when stored at ) 80 °C in their storage buffer. AK3 proved to be highly unstable when stored at room temperature, losing 95% of its activity in 24 h. During this period of time, AK1 and AK2 retained all their activity. Kinetic parameters The three enzymes displayed hyperbolic kinetics with both ATP and Asp (not shown). The AK1 catalytic constant (k cat ) was two-fold higher than the AK2 k cat and three-fold higher than the AK3 k cat (Table 1). The AK3 K m for ATP was about two-fold and three-fold lower than observed for AK2 and AK1, respectively. The AK3 K m for Asp was about two-fold lower than observed for AK1 and AK2 (Table 1). In agreement with a random mechanism for AK [7], the apparent K m Fig. 1. Protein purification. Proteins were separated on a 10% poly- acrylamide (w ⁄ v) slab gel under denaturing conditions and stained with Coomassie brilliant blue R-250. Lane 1: molecular mass mark- ers. Lanes 2, 4 and 5: soluble proteins of the E. coli extract contain- ing AK1, AK2 and AK3, respectively. Lanes 3, 5 and 6: purified AK1, AK2 and AK3, respectively (1 lg). Table 1. Kinetic parameters. Activities were measured in 50 mM Hepes (pH 8.0), 150 mM KCl, 20 mM MgCl 2 and 200 lM NADPH with 100 n M AK and 1 lM aspartate semialdehyde dehydrogenase at 30 °C. K mATP (lM) K mAsp (lM) k cat (s )1 ) AK1 1700 (±190) 2037 (±90) 23.4 ± 0.5 AK2 980 (±70) 1940 (±215) 14.5 ± 0.6 AK3 560 (±40) 1095 (±45) 8.4 ± 0.1 G. Curien et al. A. thaliana monofunctional aspartate kinases FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS 165 for one substrate was independent of the concentration of the other substrate in the absence of inhibitor. Regulatory properties Regulatory properties were subsequently examined in the presence of physiologic concentrations of Asp (1 mm) and ATP (2 mm). The results shown in Fig. 2 show that the enzymes were inhibited in a sigmoidal manner by increasing concentrations of Lys. AK1 dis- played a much higher K 0.5 value for Lys inhibition (570 ± 20 lm; Fig. 2A) compared to AK2 (K 0.5 ¼ 10.2 ± 0.7 lm; Fig. 2B) and AK3 (K 0.5 ¼ 7.4 ± 0.4 lm; Fig. 2C). For the three enzymes, no inhibition by SAM could be detected in the absence of Lys. Interestingly, in the presence of Lys, AK1 but not AK2 or AK3 was inhibited by SAM (Fig. 3). As shown in Fig. 3A, for AK1 the K 0.5 value for Lys inhi- bition decreased dramatically from 570 lm in the absence of SAM to 4.2 ± 0.2 lm in the presence of a saturating concentration of SAM (300 lm). Thus, at saturation with SAM, AK1 displayed a K 0.5 value for Lys similar to that of the SAM-insensitive AK2 and AK3 enzymes. The response curves of AK1 to SAM in the presence of Lys are shown in Fig. 3B,C. In the presence of Lys, AK1 activity decreased in a sigmoidal manner as a function of SAM concentration. Increasing the Lys concentration decreased the K 0.5 values for SAM (15 lm in the presence of 100 lm Lys). Nature of the inhibition In order to determine the origin of the inhibition, AK activities were measured in the presence of variable concentrations of ATP and Asp for different concen- trations of Lys (as well as in the presence of Lys plus SAM for AK1). The results are shown in Fig. 4 for AK1 and in Fig. 5 for AK2 (qualitatively identical results were obtained with AK3; not shown). In the presence of Lys, the apparent K m values for both ATP and Asp increased. Note that the increase in the apparent K m values for the substrates in the presence of the inhibitors was more pronounced at low concen- trations of the second substrate. Thus, whereas in the absence of inhibitor, the apparent K m for one substrate was independent of the concentration of the other sub- strate (empty squares for AK1; Fig. 4A,B) and empty circles for AK2; Fig. 5A,B), a dependence was observed in the presence of the inhibitor. Increasing the second substrate concentration decreased the K m effect of Lys. This effect is expected when competitive inhibition occurs with a two-substrate enzyme. The same behavior was observed with the SAM-sensitive AK1 enzyme in the presence of Lys plus SAM. The two-substrate nature of AKs necessitates caution in the examination of the inhibitor effect on catalytic constant. At subsaturating (i.e. limiting) fixed concen- tration of ATP (or Asp) and variable concentrations of Asp (or ATP), the maximal v ⁄ [E] 0 value is an apparent catalytic constant. Increasing the inhibitor concentration increases K m for both the fixed and the variable substrates, thus leading to a decrease in apparent maximal velocity. In order to check whether 0 0.2 0.4 0.6 0.8 1 0 500 1000 1500 2000 KA1a citvity (R letaiv u enits) [Lysine], µM A AK1 0 0.2 0.4 0.6 0.8 1 020406080100 AK ca 2tiivty leR(ativeinu ts) [Lysine], µM AK2 B 0 0.2 0.4 0.6 0.8 1 01020304050 AK a 3ctiivty (R letaiveu nits) [L y sine], µ M AK3 C Fig. 2. Inhibition of Arabidopsis monofunctional AK isoenzymes by Lys. AK activities were measured in buffer D in the presence of 1m M Asp, 2 mM ATP and variable concentrations of Lys. Activities were normalized to unity in the absence of inhibitors. Curves are the best fit obtained by nonlinear regression analysis of the experi- mental data using a Hill equation. (A) AK1 (j), (B) AK2 (s) and (C) AK3 (n) activities versus Lys concentration. Values for AK1 are: K 0.5 ¼ 570 ± 10 lM, n H ¼ 2.4 ± 0.1. Values for AK2 are: K 0.5 ¼ 12.5 ± 0.8 l M, n H ¼ 1.3 ± 0.1. Values for AK3 are: K 0.5 ¼ 7.4 ± 0.2 l M, n H ¼ 2.6 (± 0.2). A. thaliana monofunctional aspartate kinases G. Curien et al. 166 FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS the true catalytic constant was affected by Lys, extra- polated apparent catalytic constants obtained by curve-fitting in bisubstrate variation experiments were replotted as a function of the fixed substrate concen- tration (ATP or Asp). As shown in Figs 4C,D,G,H and 5C,D, extrapolated maximal v ⁄ [E] 0 values (i.e. true catalytic constants) were about 25 s )1 for AK1 and 14 s )1 for AK2. These values are similar, within the limits of experimental error, to the true catalytic con- stant of the uninhibited enzymes (Table 1). Thus, inhi- bition of plant AKs results only from a modification of the apparent K m for the two substrates ATP and Asp. Equilibrium binding experiments Kinetic experiments indicated that SAM alone had no effect on AK1 activity (Fig. 3B), even after preincuba- tion of the enzyme with SAM. However, this did not exclude SAM binding to the free enzyme without hav- ing any effect on the enzyme activity in the absence of Lys. In order to determine whether SAM binds to the free enzyme, equilibrium binding experiments were car- ried out with S-adenosyl-l-[methyl- 3 H]methionine. As shown in Fig. 6, bound radioactivity was undetectable in the absence of Lys. On the contrary, in the presence of 1 mm Lys, bound radioactivity was detected. Lys alone was able to inhibit AK1 (Fig. 2A), indicating that it can bind to the enzyme in the absence of SAM. Thus, kinetics and equilibrium binding experiments show that SAM and Lys binding to AK1 is sequential, with Lys binding preceding SAM binding. Slow-inhibition kinetics AK1 inhibition by Lys alone was fast. Indeed, a steady-state rate was achieved during the mixing time (less than 6 s; Fig. 7A), indicating rapid equilibration of AK1, and AK1–Lys and AK1–substrate complexes. In marked contrast, when AK1 activity was measured in the simultaneous presence of SAM and Lys, a delay was observed in attainment of the steady state. When the reaction was initiated with the enzyme (Fig. 7B), rates decreased with time until the steady state was reached. When, instead, the enzyme was preincubated for 5 min with the two inhibitors and diluted with the inhibitors in the reaction mix, progressive reactivation of the enzyme was observed, indicating that the inhibi- tion was reversible (not shown). Slow inhibition of plant AK has never been described before, probably because the low abundance of the enzyme in extracts from plants required long incubation times [1,2]. Three mechanisms have been proposed for the analysis of slow-binding inhibition [8,9]. Mechanism A assumes that the formation of an enzyme–inhibitor (EI) com- plex is a single, slow step. Mechanism B assumes the rapid formation of an EI complex that then undergoes 0 0.2 0.4 0.6 0.8 1 0 500 1000 1500 2000 1KAca itv yti (Relat vieu ni st) [Lys], µM A 0 µM 20 µM 300 µM [SAM] 0 0.2 0.4 0.6 0.8 1 0 100 200 300 400 1 KAca tiv yti (R vitaleu ensti) [SAM], µM B 10 µ M Lys 0 0.2 0.4 0.6 0.8 1 0255075100 1KAca tiv yti (R vitaleu ensti) [SAM], µ M C 100 µM Lys Fig. 3. Synergistic inhibition of AK1 by Lys and SAM. AK1 activity was measured in buffer D in the presence of 1 m M Asp, 2 mM ATP and variable concentrations of Lys and SAM. (A) AK1 activity versus Lys concentration for three different concentrations of SAM (0, 20, 300 l M). Curves are the best fit obtained by nonlinear regression analysis of the experimental data using a Hill equation. K 0.5 values for Lys in the presence of 0, 20 and 300 l M SAM are 570 ± 10 lM, 82 ± 2 l M, and 4.5 ± 0.5 lM, respectively. Hill numbers (n H ) were 2.4 ± 0.2, 2.3 ± 0.1, and 2 ± 0.3, respectively. (B) AK1 activity ver- sus SAM concentration in the absence and the presence of 10 l M Lys. The curve in the presence of 10 lM Lys is the best fit obtained by nonlinear regression analysis of the experimental data using a Hill equation. The K 0.5 value for SAM in the presence of 10 lM Lys is 131 ± 6 l M. The Hill number in the presence of 10 lM Lys is 1.75 ± 0.1. (C) AK1 activity versus SAM concentration in the pres- ence of 100 l M Lys. The K 0.5 value is 15 ± 0.3 lM. The Hill number is 2 ± 0.05. G. Curien et al. A. thaliana monofunctional aspartate kinases FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS 167 0 1 2 3 4 5 024681012 K m pap of rA TP m( M ) [Asp], mM A 0 6 12 18 24 30 0510152 k act pa p s( 1- ) [Asp], mM C 0 1 2 3 4 0 5 10 15 20 25 K m pap ofrA TP m( M ) [Asp], mM E [Lys]=3 µM [SAM]=400 µM 0 6 12 18 24 30 0 5 10 15 20 k ac t pap s( 1- ) [As p ], m M [Lys]= 3 µ M [SAM]= 400 µM G 2 3 4 5 6 7 024681 K m ppa f oA rs Mm( p) [ATP], m M B 0 0 6 12 18 24 30 02468 k a c t pap s( 1- ) [ATP], m M D 0 2 4 6 024681 K m pa p of rA sp m( M) [ATP], m M [Lys ]= 3 µ M [SAM] = 400 µ M F 0 0 6 12 18 24 30 02468 k act p ap s ( 1 - ) [ATP], m M [Lys]= 3 µ M [SAM] = 400 µ M H Fig. 4. Nature of the inhibition of AK1 by Lys or Lys plus SAM. Bisubstrate variation experiments were carried out with AK1 in the absence of inhibitor (h), in the presence of Lys alone (500 l M)(j) or in the presence of Lys (3 lM) plus SAM (400 lM)( ). Hyperbolic curves (not shown) obtained for a fixed concentration of one substrate and variable concentrations of the other substrate were fitted with Michaelis–Menten equations to calculate apparent kinetic parameters (K app m and k app cat ). (A) AK1 apparent K m for Asp versus ATP concentration in the absence (h) and presence (j) of 500 l M Lys. (B) AK1 apparent K m for ATP versus Asp concentration in the absence (h) and presence (j) of 500 l M Lys. (C) AK1 apparent k cat values (extrapolated from bisubstrate variation experiments) versus Asp concentration for 500 l M Lys. (D) AK1 extrapolated apparent k cat values versus ATP concentration in the presence of 500 l M Lys. The extrapolated maximal apparent k cat value is similar (within the limits of experimental error) to the true k cat value of AK1, indicated by a dotted line (Table 1). (E, F, G, H) Same as (A), (B), (C) (D), respectively, for bisubstrate variation experiments carried out with AK1 and in the presence of Lys plus SAM. A. thaliana monofunctional aspartate kinases G. Curien et al. 168 FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS a slow and favorable isomerization to an EI* complex. In the mechanism C, isomerization precedes inhibitor binding. It is possible to distinguish between these mechanisms by analysis of the relationship between the exponential decay constant (k obs ) of the progress curve and the inhibitor concentration [8,9]. A linear relation- ship is observed for mechanism A, and hyperbolic rela- tionships for mechanisms B and C: however, k obs increases with inhibitor concentration when mechan- ism B applies, and decreases when mechanism C applies. Concerning AK1, kinetic results indicate that Lys alone inhibits the enzyme (although with a low apparent affinity) and that the inhibition is fast. Also, equilibrium binding analyses indicate that SAM bind- ing follows Lys binding. Slow inhibition in the pres- ence of SAM results either from slow binding of SAM to the AK1–Lys complex (mechanism A), or from a slow conformational transition of the AK1– Lys–SAM complex induced by SAM (mechanism B), or finally, from the binding of SAM to an isomer of the enzyme–Lys complex in slow equilibrium with another isomer (mechanism C). In order to distin- guish between the three possible mechanisms, progress curves were obtained with AK1 for 100 lm Lys (i.e. under conditions where Lys alone was only margin- ally inhibitory) and for different concentrations of SAM. The k obs values were obtained by nonlinear least-square fitting of the progress curves using Eqn (1): A t ¼ A t 0 À v s Á t þ ðv s À v i ÞÁð1 À e Àk obs t Þ k obs ð1Þ where A t is the absorbance at time t, A t0 is the absorb- ance at t 0 , v i is the initial velocity of the reaction, v s is the steady-state velocity of the reaction, and k obs is an exponential decay constant. The results shown in Fig. 7C reveal a hyperbolic relationship between k obs and SAM concentration. The k obs value increased when SAM concentration increased. The results are thus consistent with mechan- ism B, i.e. a mechanism in which slow inhibition is due to slow isomerization following SAM binding to an enzyme–Lys complex. 0 1 2 3 01234 K m pa p ofrA TP m( M ) [Asp], mM A 0 5 10 15 20 0123 k act pap s( 1- ) [Asp], mM C 4 0 2 4 6 8 01234 K m a pp ofrA sp m( M) [ATP], mM B 0 5 10 15 20 01234 k tac app ( s 1- ) [ATP], mM D Fig. 5. Nature of the inhibition of AK2 by Lys. (A) AK2 apparent K m for Asp versus ATP concentration in the absence (s) or in the presence (d)of25l M Lys. (B) AK2 apparent K m for ATP versus Asp concentration in the absence (s) or in the presence (d)of25lM Lys. (C) AK2 extrapolated apparent k cat values (calculated from bisubstrate variation experiments) versus Asp concentration in the presence of 25 lM Lys. The maximal value is similar to the true AK2 k cat value, symbolized by a dotted line (Table 1). (D) AK2 extrapolated apparent k cat values (cal- culated from bisubstrate variation experiments) versus ATP concentration in the presence of 25 l M Lys. The maximal value is similar to the true AK2 k cat value (Table 1). G. Curien et al. A. thaliana monofunctional aspartate kinases FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS 169 AK2 and AK3 also displayed slow inhibition kinet- ics by Lys (not shown). In the absence of structural data for these enzymes, the results cannot yet be inter- preted in terms of a mechanistic model. Allosteric control of monofunctional AKs is highly specific No additional inhibition of the three AKs was observed upon addition of Thr or Leu, in the presence of 5 lm Lys for AK3 and AK2, and in the presence of 100 lm Lys and 20 lm SAM for AK1. This contrasts with E. coli AKIII, which proved to be inhibited synergistically by Lys and Leu [10], and Bacillus polymyxa monofunctional AK, which is inhibited in a concerted manner by Lys and Thr [11]. In addition, the activators of Thr-sensitive bifunctional AK–homo- serine dehydrogenases from plants (Ala, Cys, Ile, Leu, Ser and Val [3]) had no effect on monofunctional A. thaliana AK activities, either in the absence or in the presence of the inhibitors. The other amino acids tested (Met, Gln, Asn, Glu, Arg) had no effect on the enzyme activities at 2.5 mm either in the presence or the absence of the inhibitor Lys or Lys plus SAM (for AK1). Discussion The present article describes in detail the kinetic and regulatory properties of the three Lys-sensitive mono- functional AKs from Arabidopsis. We show for the first time that all three enzymes are inhibited by Lys, but only one isoform (AK1) is inhibited synergistically by Lys and SAM. In vitro kinetic measurements indi- cate that all three enzymes are efficiently inhibited by physiologic concentrations of Lys (80 lm). Indeed, the K 0.5 values are 80 lm,10lm and 7 lm, for AK1, AK2 and AK3, respectively, for activity assayed in the pres- ence of physiologic concentrations of Asp, ATP and SAM (for AK1). AK2 and AK3 would be more strongly inhibited by Lys than AK1 under these condi- tions. AK1 activity is also highly sensitive to changes in SAM concentrations in the physiologic context of the leaf (K 0.5 for SAM in the presence of 80 lm Lys is close to the physiologic concentration of SAM, 20 lm 0 0.25 0.5 0.75 1 0 20 40 60 80 100 SAM ep romno rem [SAM], µ M Fig. 6. Equilibrium binding of S-adenosyl-L-[methyl- 3 H] methionine to AK1 in the absence (h) and in the presence (j)of1m M Lys. The curve in the presence of 1 m M Lys is the best fit obtained by nonlin- ear regression analysis of the experimental data using the equation of a hyperbol. A K d value of 5.7 ± 0.7 lM could be calculated. 0.9 1 0 100 200 300 400 time (s) A [Lys] = 500 µM No SAM 0.6 0.7 0.8 0.9 1 050100150200 A 43mn 0 A 43mn 0 time (s) [SAM] (µ M) 400 200 100 50 [Lys]= 100 µ M B 0 0 0.015 0.03 0.045 0.06 0 100 200 300 400 500 k sbo s( 1- ) [SAM], µ M [Lysine]= 100 µM C Fig. 7. Slow inhibition of AK1 in the presence of Lys plus SAM. (A) Progress curves were obtained in the presence of Lys and in the absence of SAM, with the reaction initiated with AK1. (B) Progress curves were obtained in the presence of Lys and SAM in the reac- tion media. Reactions were initiated with AK1 free of inhibitors. (C) Observable rate constant of AK1 versus SAM concentration. Pro- gress curves were obtained as indicated in (B) for 100 l M Lys and different concentrations of SAM. For each curve, the exponential decay constant was obtained by curve-fitting using Eqn (1). Experi- ments were repeated twice for a given SAM concentration. Data points were fitted with the equation derived in [6] for mechanism B (two-step process). A. thaliana monofunctional aspartate kinases G. Curien et al. 170 FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS [12]). It is clear from our results that AKs are not sat- urated by the substrates in vivo. Thus, control of plant AK activities by modification of the enzyme K m values for the substrates is an efficient control mechanism. Although the Thr-sensitive bifunctional AKs [3] and Lys-sensitive monofunctional AKs from Arabidopsis have in common a control of their activity via modifi- cation of the K m values for both substrates, a striking difference is the absence of activation of monofunc- tional AKs by small amino acids (Ala, Cys, Ile, Leu, Ser, Val). Interestingly, monofunctional AKs display low K m values for Asp and ATP (1–2 mm in the absence of inhibitors) compared to bifunctional AKs (5–15 mm in the absence of the activators). The activa- tors of bifunctional AKs reduce the K m values for both ATP and Asp to values similar to those measured here for monofunctional AKs, suggesting that under physi- ologic conditions, all five AKs display similar kinetic efficiencies. We proposed that Ala, because of its abundance in the chloroplast, was the physiologic acti- vator of bifunctional AKs. A hypothetical functional advantage of this allosteric interaction could be a feed- forward control, coupling the Asp-derived amino acid pathway to nitrogen and carbon metabolism. Accord- ing to this hypothesis, one would expect to also observe allosteric activation of Lys-sensitive AKs. Its absence suggests that the effect of activation of Thr- sensitive bifunctional AKI and AKII might be to increase their sensitivity to Thr inhibition rather than to provide coupling with carbon and nitrogen metabo- lism. This might also explain why the activation of AKI and AKII need not be highly specific for the acti- vator [3]. A survey of the expression pattern of the AK genes using the Arabidopsis microarray database Genevesti- gator [13] indicated the presence of AK1 and AK3 mRNAs in all the examined organs and tissues at sim- ilar levels. Specific expression of the AK3 gene in vas- cular tissues has been reported [14]. As the phenotype of a knockout mutant of AK3 [14] is indistinguishable from that of a wild-type Arabidopsis, other AKs (more probably the closely related AK2) can replace AK3, at least under controlled growth conditions. No data could be found for the AK2 gene in the Arabidopsis microarray database Genevestigator. However, nor- thern blot analyses [15] suggested that the gene enco- ding AK2 is expressed in all tissues of Arabidopsis. Unless specific control of translation takes place in vivo, these results suggest that the three AKs are coexpressed in leaf chloroplasts. Together with our kinetic results, they suggest that a fraction of the flux controlled by Lys is insensitive to SAM (i.e. the flux generated by the activity of AK2 and AK3). Mechanism of inhibition of AK1 by Lys and SAM The mechanism of the synergistic inhibition of AK1 by Lys and SAM was analyzed according to the recent three-dimensional structure of the AK1–Lys–SAM complex in this laboratory [4]. AK1 displayed a K 0.5 value for Lys inhibition in the absence of SAM about 50-fold higher than that observed for the SAM-insen- sitive AKs. This might be due to a much higher affin- ity of AK1 for the substrates compared to AK2 and AK3. That is, much more Lys might be required to displace more strongly bound substrates. However, all three AKs display similar K m values for the substrates (Table 1). Thus, the high AK1 K 0.5 value for Lys inhi- bition in the absence of SAM (Fig. 2A) is a conse- quence of the enzyme’s lower affinity for Lys compared to AK2 and AK3. Equilibrium binding analyses carried out with AK1 indicated sequential binding of Lys and then SAM to AK1 (Fig. 6). In the crystal structure of AK1 in com- plex with Lys and SAM, the SAM-binding site in the regulatory domain of the enzyme is formed in part by a loop that also participates in the Lys-binding site. This suggests that the SAM-binding site is not already pre- sent on the enzyme and requires prior binding of Lys. In the presence of SAM, the apparent affinity of AK1 for Lys was much higher than in its absence and was similar to that of the SAM-insensitive AK2 and AK3. This increase in apparent affinity in the presence of SAM does not result from a direct molecule-to- molecule interaction between SAM and Lys, but is mediated by the protein. Indeed, the Lys- and SAM- binding sites are in close proximity in the crystal struc- ture, and two adjacent amino acids of the polypeptide chain, S371 and V372, interact with Lys and with Lys and SAM, respectively [4]. The increase in apparent affinity of AK1 for Lys in the presence of SAM results from a slow conformational rearrangement of the pro- tein that is induced by SAM, as indicated by the hyperbolic relationship between k obs and SAM concen- tration (Fig. 7C). The data can be used to propose a model for the inhibition of AK1 by Lys and SAM (Scheme 1). In Scheme 1, E represents the enzyme in the active con- formation. ES represents the enzyme–substrate (ATP plus Asp) complex. In this model, upon Lys binding in the regulatory domain, the enzyme adopts a novel con- formation (E*) that is unable to bind the substrates (Lys alone is inhibitory; Fig. 2A). The transition E–Lys fi E*–Lys is fast (Fig. 7A), but the equilib- rium is not favorable. That is, a high concentration of Lys is required to shift the whole enzyme population to the inhibited form. In addition, the E*–Lys form G. Curien et al. A. thaliana monofunctional aspartate kinases FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS 171 has acquired the ability to bind SAM (Fig. 6). Follow- ing the formation of the encounter complex with SAM (SAM–E*–Lys), a slow conformational transition induced by SAM occurs (Fig. 7C). As shown in the protein structure [4], Lys is trapped inside the protein in the E** state. The ribose and adenine moieties of SAM are also deeply buried in the protein, but the Met moiety of SAM is exposed to the solvent. Most probably, the dissociation of the SAM–E**–Lys com- plex occurs when the protein complex is in the SAM– E*–Lys conformational state. The release of the coin- hibitors is sequential, with Lys release following SAM release. In this model, the reinforcement of Lys inhibi- tion by SAM would result from the displacement of the unfavorable equilibrium between E–Lys and E*– Lys, owing to the formation of an enzyme form stabil- ized by SAM. The sigmoidal shape of the Lys inhibition curves (Fig. 2A) is in accordance with the identification of two equivalent Lys-binding sites at the interface formed by two regulatory domains in the protein dimer [4]. Residues from both monomers contribute to the formation of a Lys-binding site, thus providing strong coupling between both subunits. In the crystal structure, the number of interactions between dimers is low (eight hydrogen bonds, 2.4% of each subunit area), in agreement with native gel electrophoresis results showing that the enzyme behaves predomin- antly as a dimeric enzyme (95%) in equilibrium with a tetramer. However, kinetic experiments indicate that the Hill numbers for AK1 are close to or slightly higher than 2. This may indicate that a fraction of the enzyme population forms tetramers in solution under the conditions of the kinetic experiments. Binding experiments indicated hyperbolic saturation curves for SAM in the presence of 1 mm Lys (Fig. 6). This suggests that there are no cooperative homotropic interactions between the two SAM molecules in the protein dimer under these conditions. This is supported by the three-dimensional structure. The SAM-binding site of a monomer is entirely formed by amino acids from that monomer, with no obvious physical inter- actions between this site and the other subunit. Kinetic experiments showed sigmoidal SAM saturation curves (Fig. 3B,C). This may indicate that long-range interac- tions occur in the enzyme between SAM sites. However, as SAM binding follows Lys binding, the cooperative homotropic interaction observed for SAM under these conditions may be only apparent and result from homo- tropic interactions between Lys-binding sites. As discussed by Mas-Droux et al. [4], differences in amino acid sequences were observed in AK2 and AK3 compared to AK1 at the level of the SAM-binding site. W392SR394 amino acids are involved in SAM binding in AK1. The tryptophan residue is not found in AK2, and the loop is longer in AK3. These differ- ences could explain the absence of SAM effects on AK2 and AK3. Comparison of plant and bacterial Lys-sensitive AKs In addition to the specific control of AK1 by Lys plus SAM, Arabidopsis AK allosteric control displayed dif- ferences compared to the E. coli Lys-sensitive AKIII enzyme. First, E. coli AKIII is inhibited in synergy by Lys and Leu [10], but no effect of Leu could be observed with the A. thaliana monofunctional AKs. In addition, the three A. thaliana AKs display slow-inhi- bition kinetics, a feature that has never been reported for the bacterial AKIII enzyme. Finally, inhibition of A. thaliana enzymes results exclusively from a modifi- cation of the apparent K m for the substrates ATP and Asp. Three studies examined the inhibition pattern of E. coli AKIII by Lys [16–18]. All report competitive inhibition with respect to ATP (as for the plant enzyme; this work) but noncompetitive inhibition with respect to Asp [16,17]. However, in these studies, the effect of Lys on E. coli AKIII was tested with high concentration of the second substrate ATP. In these conditions, the K m effect may have been minimized (see Figs 4A,B and 5A,B for A. thaliana AK). Lower concentrations of ATP were used by Wampler & West- head [18], and the apparent K m for Asp increased from 1.6 mm in the absence of Lys to 5 mm at 560 lm Lys, suggesting a competitive component in the inhibition by Lys. In the same study, the authors reported a modification of maximal velocity in the presence of Lys for a fixed concentration of ATP and variable con- centrations of Asp. However, as the ATP concentra- tion was fixed, they probably observed a decrease in apparent maximal velocity (a consequence of a E syL-E syL-*E SE P+E syL-*E-MAS syL-* * E-MAS wolS Scheme 1. A. thaliana monofunctional aspartate kinases G. Curien et al. 172 FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS decrease in the apparent affinity for the second sub- strate ATP used at a fixed concentration) rather than a decrease in the true maximal velocity (true k cat ). If this is correct, then plant AK and bacterial AK inhibition mechanisms may be similar. The publication of the AK1–Lys–SAM complex structure was followed by the release of the Methanococ- cus jannaschii AK structure in complex with Mg-ADP and Asp [19] (Protein Data Bank entry 2HMF) and two structures of the E. coli AKIII Lys-sensitive enzyme (AKIII–Lys–Asp and AKIII–Asp–ADP complexes; Protein Data Bank entries 2J0X and 2J0W, respectively) [20]. This offers the possibility of examining the inhibi- tion mechanisms of the plant and the bacterial enzymes in the light of structural data. The three-dimensional structure of AK1 in complex with Lys and SAM [4] revealed that the conformation of the ATP-binding site in this complex was unsuitable for nucleotide binding. In E. coli AKIII cocrystallized with Lys, the ATP-bind- ing site was also in a conformation that prevented the accommodation of ATP. This is consistent with compet- itive inhibition with respect to ATP observed both with plant AKs and with E. coli AKIII [16–18]. The active site arginine side chain (R198 in M. jann- aschii AK and R202 in E. coli AKIII) was shown to be responsible for a bidendate interaction with the Asp substrate a-carboxyl group [19]. The side chain of the corresponding Arg residue (R230) in the A. thaliana AK1–Lys–SAM complex is farther away (7 A ˚ ) and forms interactions with the SAM-binding site, suggest- ing that the binding of the inhibitors removes an inter- action that stabilizes Asp in its binding site. This is in agreement with the observed increase in K m values for Asp in the presence of the inhibitor(s). In E. coli AKI- II cocrystallized with Lys, an Asp molecule was pre- sent in the active site (one per dimer) [20]. In this complex, the active site Arg residue (R202) is posi- tioned more than 2 A ˚ further from the Asp substrate molecule than in the AKIII–Asp–ADP complex. This suggests that the interaction between the AKIII active site and the Asp substrate is weaker in the inhibited complex. This is in agreement with an increase in the apparent K m for Asp observed in the presence of Lys, as reported by Wampler & Westhead [18]. Structural data thus suggest that the plant and the E. coli enzymes are controlled by similar mechanisms. The E. coli Lys-sensitive AK has been considered by Monod et al. [21] and others [10,22] to be a V-type allosteric enzyme. Both plant and E. coli AKs indeed display features of allosteric V systems, as substrate saturation curves are hyperbolic in the absence and in the presence of the effector. However, in the model proposed by Monod et al. for ‘V systems’, the alloster- ic effector modifies k cat and the substrate has the same affinity for the two states of the enzyme (the R and T states). It would be somewhat misleading to consider plant AKs as V-type allosteric enzymes, as Lys modi- fies exclusively the apparent K m for the substrates. The denomination ‘V system’ and the associated model may not be appropriate for this enzyme. Inhibition and synergistic inhibition of AKs Synergistic control by Lys and SAM is specific to the plant enzyme, but other AKs also display synergistic inhibition. Lys-sensitive AKIII from E. coli is inhibited in synergy by Lys and Leu [22]. AKIII from Bacillus subtilis [23,24] and AK from Rhodopseudomon- as [25,26], display synergistic inhibition by Lys and Thr. Synergistic inhibition requires the existence in these enzymes of two sites, one for each coinhibitor. The reg- ulatory domain of A. thalian a AK1 as well as that of E. coli AKIII is formed of two ACT domains [27]. In AK1 from Arabidopsis, the two coinhibitors Lys and SAM bind in one of the two ACT domains (ACT1) [4]. In the E. coli AKIII structure, the Lys molecule was also found in ACT1 [20]. The enzyme structure in the presence of Lys plus Leu is still unknown. Thus we do not know whether the coinhibitors in the other synergis- tically inhibited AKs bind in a position similar to where the SAM molecule binds in AK1 or whether another site (in ACT2, for example) is involved in the binding of the coinhibitor. According to the first hypothesis, AK1 may provide an explanatory model for the other AKs that are synergistically inhibited. Experimental procedures Chemicals Amino acids were obtained from Sigma-Aldrich (St Quentin Fallavier, France). SAM was purified as previously described [28]. Bacterial strains Escherichia coli strain DH10B was used for cloning, and E. coli strain BL21 (DE3) pLysS codon+ (Novagen, Darmstadt, Germany) was used for recombinant protein production. Construction of the plasmids The cDNA sequences corresponding to the predicted mature proteins were amplified by PCR using an A. thali- ana cDNA library [29]. The 5¢ and 3¢ oligonucleotides G. Curien et al. A. thaliana monofunctional aspartate kinases FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS 173 [...]... Identification of six novel allosteric effectors of Arabidopsis thaliana aspartate kinase–homoserine dehydrogenase isoforms: physiological context sets the specificity J Biol Chem 280, 41178–41183 4 Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase Plant... methionine and threonine biosynthesis pathways in Arabidopsis thaliana Eur J Biochem 270, 4615–4627 13 Zimmermann P, Hirsch-Hoffmann M, Hennig L & Gruissem W (2004) GENEVESTIGATOR Arabidopsis microarray database and analysis toolbox Plant Physiol 136, 2621–2632 14 Yoshioka Y, Kurei S & Machida Y (2001) Identification of a monofunctional aspartate kinase gene of Arabidopsis thaliana with spatially and temporally... localisation of aspartate kinase and the enzymes of threonine and methionine biosynthesis in green leaves Plant Physiol 71, 780–784 6 The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana Nature 408, 796–815 7 Angeles TS & Viola RE (1990) The kinetic mechanisms of the bifunctional enzyme aspartokinase–homoserine dehydrogenase I from Escherichia... monofunctional aspartate kinases 8 Morrison JF & Walsh CT (1988) The behavior and significance of slow-binding enzyme inhibitors Adv Enzymol Relat Areas Mol Biol 61, 201–301 9 Szedlacsek SE & Duggleby RG (1995) Kinetics of slow and tight-binding inhibitors Methods Enzymol 249, 144–180 10 Mazat JP & Patte JC (1976) Lysine-sensitive aspartokinase of Escherichia coli K12 Synergy and autosynergy in an allosteric. .. with a coupled assay using aspartate semialdehyde dehydrogenase from A thaliana [32] NADPH disappearance was monitored at 340 nm in a thermostated quartz cuvette (100 lL) using a Uvikon 930 (Bioteck Instruments, Neufahrn, Germany) spectrophotometer The amount of aspartate semialdehyde dehydrogenase was adjusted so that the flux through the two-enzyme system was not dependent on aspartate semialdehyde dehydrogenase... Acta 113, 531–541 18 Wampler DE & Westhead EW (1968) Two aspartokinases from Escherichia coli Nature of the inhibition and molecular changes accompanying reversible inactivation Biochemistry 7, 1661–1671 19 Faehnle CR, Liu XY, Pavlovsky A & Viola RE (2006) The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase Acta Crystallogr F 62, 962–966 20 Kotaka... thaliana with spatially and temporally regulated expression Genes Genet Syst 76, 189–198 15 Tang G, Zhu-Shimoni JX, Amir R, Zchori IB & Galili G (1997) Cloning and expression of an Arabidopsis thaliana cDNA encoding a monofunctional aspartate kinase homologous to the lysine-sensitive enzyme of Escherichia coli Plant Mol Biol 34, 287–293 16 Stadtman ER, Cohen GN, Lebras G & De-RobichonSzulmajster H (1961)...A thaliana monofunctional aspartate kinases G Curien et al contained an NcoI site and XhoI site, respectively The resulting PCR fragments were digested by NcoI and XhoI and ligated into pET 23d(+) vector (Novagen) All proteins... JP, Felenbok B & Patte JC (1974) The role of lysine and leucine binding on the catalytical FEBS Journal 274 (2007) 164–176 ª 2006 The Authors Journal compilation ª 2006 FEBS 175 A thaliana monofunctional aspartate kinases 23 24 25 26 27 28 G Curien et al and structural properties of aspartokinase III of Escherichia coli K 12 Eur J Biochem 48, 147–156 Paulus H & Gray E (1964) Multivalent feedback inhibition... 112, 251–258 Hitchcock MH & Hodgson B (1976) Lysine- and lysineplus-threonine-inhibitable aspartokinases in Bacillus brevis Biochim Biophys Acta 445, 350–363 Chipman DM & Shaanan B (2001) The ACT domain family Curr Opin Struct Biol 11, 694–700 Curien G, Job D, Douce R & Dumas R (1998) Allosteric activation of Arabidopsis threonine synthase by S-adenosylmethionine Biochemistry 37, 13212– 13221 176 29 Elledge . Allosteric monofunctional aspartate kinases from Arabidopsis Gilles Curien, Mathieu Laurencin, Myle ` ne Robert-Genthon. genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited. 2006) doi:10.1111/j.1742-4658.2006.05573.x Plant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains