Use of Biomolecular Interaction Analysis to Elucidate the Regulatory Mechanism of the Cysteine Synthase Complex fromArabidopsis thaliana
2002; Elsevier BV; Volume: 277; Issue: 34 Linguagem: Inglês
10.1074/jbc.m111632200
ISSN1083-351X
AutoresOliver Berkowitz, Markus Wirtz, Alexander Wolf, Jürgen Kuhlmann, Rüdiger Hell,
Tópico(s)Lipid metabolism and biosynthesis
ResumoReal time biomolecular interaction analysis based on surface plasmon resonance has been proven useful for studying protein-protein interaction but has not been extended so far to investigate enzyme-enzyme interactions, especially as pertaining to regulation of metabolic activity. We have applied BIAcore technology to study the regulation of enzyme-enzyme interaction during mitochondrial cysteine biosynthesis in Arabidopsis thaliana. The association of the two enzyme subunits in the hetero-oligomeric cysteine synthase complex was investigated with respect to the reaction intermediate and putative effector O-acetylserine. We have determined an equilibrium dissociation constant of the cysteine synthase complex (KD = 25 ± 4 × 10−9m), based on a reliable A + B ⇔ AB model of interaction. Analysis of dissociation kinetics in the presence of O-acetylserine revealed a half-maximal dissociation rate at 77 ± 4 μmO-acetylserine and strong positive cooperativity for complex dissociation. The equilibrium of interaction was determined using an enzyme activity-based approach and yielded aKm value of 58 ± 7 μmO-acetylserine. Both effector concentrations are in the range of intracellular O-acetylserine fluctuations and support a functional model that integrates effector-driven cysteine synthase complex dissociation as a regulatory switch for the biosynthetic pathway. The results show that BIAcore technology can be applied to obtain quantitative kinetic data of a hetero-oligomeric protein complex with enzymatic and regulatory function. Real time biomolecular interaction analysis based on surface plasmon resonance has been proven useful for studying protein-protein interaction but has not been extended so far to investigate enzyme-enzyme interactions, especially as pertaining to regulation of metabolic activity. We have applied BIAcore technology to study the regulation of enzyme-enzyme interaction during mitochondrial cysteine biosynthesis in Arabidopsis thaliana. The association of the two enzyme subunits in the hetero-oligomeric cysteine synthase complex was investigated with respect to the reaction intermediate and putative effector O-acetylserine. We have determined an equilibrium dissociation constant of the cysteine synthase complex (KD = 25 ± 4 × 10−9m), based on a reliable A + B ⇔ AB model of interaction. Analysis of dissociation kinetics in the presence of O-acetylserine revealed a half-maximal dissociation rate at 77 ± 4 μmO-acetylserine and strong positive cooperativity for complex dissociation. The equilibrium of interaction was determined using an enzyme activity-based approach and yielded aKm value of 58 ± 7 μmO-acetylserine. Both effector concentrations are in the range of intracellular O-acetylserine fluctuations and support a functional model that integrates effector-driven cysteine synthase complex dissociation as a regulatory switch for the biosynthetic pathway. The results show that BIAcore technology can be applied to obtain quantitative kinetic data of a hetero-oligomeric protein complex with enzymatic and regulatory function. surface plasmon resonance cysteine synthase complex O-acetylserine O-acetylserine (thiol)-lyase serine acetyltransferase nitrilotriacetic acid relative units fresh weight The interaction of proteins is essential for the function of living cells (1Huang X. Holden H.M. Raushel F.M. Annu. Rev. Biochem. 2001; 70: 149-180Crossref PubMed Scopus (309) Google Scholar, 2Miles E.W. Rhee S. Davies D.R. J. Biol. Chem. 1999; 274: 12193-12196Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Monitoring of biomolecular interaction analysis can be achieved by BIAcore technology that is based on surface plasmon resonance (SPR).1 The physical phenomenon of SPR is widely used to visualize macromolecular interactions in real time with the advantages of no labeling requirements and the option to determine kinetic rate constants. It is mostly applied to quantification of antigen/antibody binding, receptor ligand screening, or characterization of protein modification. It has not been used so far to investigate enzyme-enzyme interactions, in particular with respect to regulation of the metabolic activity of multienzyme complexes.In this study we have applied SPR to analyze the interaction of a hetero-oligomeric metabolic protein complex using the enzymes of cysteine biosynthesis from mitochondria of Arabidopsis thaliana. Serine acetyltransferase (SAT; EC 2.3.1.30) generates the activated sulfide acceptor O-acetylserine (OAS) from serine and acetyl-CoA. O-Acetylserine (thiol)-lyase (OAS-TL; EC 4.2.99.8) then inserts free sulfide in a β-replacement reaction to synthesize cysteine and acetate. One tetramer of SAT and two dimers of OAS-TL are presumed to form the hetero-oligomeric cysteine synthase complex (CSC) that was first described for Salmonella typhimurium (3Kredich N.M. Becker M.A. Tomkins G.M. J. Biol. Chem. 1969; 244: 2428-2439Abstract Full Text PDF PubMed Google Scholar, 4Kredich N.M. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umberger E. Escherichia coli and Salmonella typhimurium. American Society for Microbiology, Washington, D. C.1996: 514-527Google Scholar). Protein-protein interactions of such metabolic enzymes mostly serve to channel substrate intermediates between active sites of sequential reaction steps of a pathway. The advantages of these quaternary organizations include efficient catalysis rates and stabilization of intermediates by prevention of losses of intermediates into solution by diffusion (1Huang X. Holden H.M. Raushel F.M. Annu. Rev. Biochem. 2001; 70: 149-180Crossref PubMed Scopus (309) Google Scholar, 2Miles E.W. Rhee S. Davies D.R. J. Biol. Chem. 1999; 274: 12193-12196Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). However, substrate channeling is not realized in the CSC, because a ready release of the reaction intermediate O-acetylserine (OAS) into the surrounding solution was observed for the bacterial complex (5Cook P.F. Wedding R.T. Arch. Biochem. Biophys. 1977; 178: 293-302Crossref PubMed Scopus (42) Google Scholar).The reason for the association of SAT and OAS-TL in the CSC is therefore unknown, despite the fact that the importance of this final step of sulfate assimilation is comparable with the fixation of ammonia by glutamine synthetase in nitrate assimilation (4Kredich N.M. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umberger E. Escherichia coli and Salmonella typhimurium. American Society for Microbiology, Washington, D. C.1996: 514-527Google Scholar, 6Saito K. Curr. Opin. Plant Biol. 2000; 3: 188-195Crossref PubMed Scopus (251) Google Scholar, 7Leustek T. Martin M.N. Bick J.-A. Davies J.P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 141-166Crossref PubMed Scopus (503) Google Scholar).In plants the synthesis of cysteine takes place in cytosol, plastids, and mitochondria. Nuclear encoded cDNAs of SAT and OAS-TL for each compartment-specific isoform have been cloned from several plants including A. thaliana (8Noji M. Inoue K. Kimura N. Gouda A. Saito K. J. Biol. Chem. 1998; 273: 32739-32745Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 9Jost R. Berkowitz O. Wirtz M. Smith L. Hawkesford M.J. Hell R. Gene (Amst.). 2000; 253: 237-247Crossref PubMed Scopus (99) Google Scholar). Expression analysis revealed semi-constitutive activity of the corresponding genes with only moderate up-regulation of mRNA steady-state levels in response to external factors such as sulfate deprivation or salt stress (10Takahashi H. Yamazaki M. Sasakura N. Watanabe A. Leustek T. de Almeida Engler J. Engler G. van Montagu M. Saito K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11102-11107Crossref PubMed Scopus (280) Google Scholar, 11Takahashi H. Watanabe-Takahashi A. Smith F.W. Blake-Kalff M. Hawkesford M.J. Saito K. Plant J. 2000; 23: 171-182Crossref PubMed Google Scholar, 12Hesse H. Lipke J. Altmann T. Höfgen R. Amino Acids (Vienna). 1999; 6: 113-131Crossref Scopus (59) Google Scholar, 13Gutierrez-Alcala G. Gotor C. Meyer A.J. Fricker M. Vega J.M. Romero L.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11108-11113Crossref PubMed Scopus (141) Google Scholar). Metabolic regulation appears to be more important for the control of flux of reduced sulfur into cysteine. Feedback inhibition by cysteine of the cytosolic SAT isoform, but cysteine insensitivity of the plastid and mitochondrial isoforms from A. thaliana, is an element of a regulatory model for cysteine synthesis (6Saito K. Curr. Opin. Plant Biol. 2000; 3: 188-195Crossref PubMed Scopus (251) Google Scholar, 8Noji M. Inoue K. Kimura N. Gouda A. Saito K. J. Biol. Chem. 1998; 273: 32739-32745Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar).An additional regulatory mechanism may be provided by the reaction intermediate OAS. As suggested by feeding experiments, OAS is involved in the de-repression of genes encoding sulfate transporters, enzymes of sulfate reduction, and a seed storage protein during sulfate deficiency (6Saito K. Curr. Opin. Plant Biol. 2000; 3: 188-195Crossref PubMed Scopus (251) Google Scholar, 14Smith F.W. Hawkesford M.J. Ealing P.M. Clarkson D.T. Vanden Berg P.J. Belcher A.R. Warrilow A.G. Plant J. 1997; 12: 875-884Crossref PubMed Scopus (238) Google Scholar, 15Kim H. Hirai M.Y. Hayashi H. Chino M. Naito S. Fujiwara T. Planta. 1999; 209: 282-289Crossref PubMed Scopus (108) Google Scholar, 16Koprivova A. Suter M. den Camp R.O. Brunold C. Kopriva S. Plant Physiol. 2000; 122: 737-746Crossref PubMed Scopus (223) Google Scholar). Furthermore, incubation of the intact CSC with OAS dissociates the complex in vitro and results in changes of kinetic properties and activity of both enzymes; free SAT tends to aggregate, becomes unstable, and loses affinity to serine and acetyl-CoA; free OAS-TL, on the contrary, gains activity and increases its affinity to OAS and sulfide (3Kredich N.M. Becker M.A. Tomkins G.M. J. Biol. Chem. 1969; 244: 2428-2439Abstract Full Text PDF PubMed Google Scholar, 17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar). These findings are corroborated by the identification of a bifunctional C-terminal domain of SAT from A. thaliana that is responsible for catalysis and protein-protein interaction with OAS-TL (18Bogdanova N. Hell R. Plant J. 1997; 11: 251-262Crossref PubMed Scopus (117) Google Scholar, 19Wirtz M. Berkowitz O. Droux M. Hell R. Eur. J. Biochem. 2001; 268: 686-693Crossref PubMed Scopus (97) Google Scholar). The low activity of OAS-TL in the CSC apparently is the reason for diffusion of OAS into the surrounding solution, thus preventing any substrate channeling (5Cook P.F. Wedding R.T. Arch. Biochem. Biophys. 1977; 178: 293-302Crossref PubMed Scopus (42) Google Scholar). An in vivo dissociation of the CSC may indeed occur, as suggested by the recent observations of increased OAS levels in response to sulfate starvation in A. thaliana (15Kim H. Hirai M.Y. Hayashi H. Chino M. Naito S. Fujiwara T. Planta. 1999; 209: 282-289Crossref PubMed Scopus (108) Google Scholar, 20Awazuhara M. Hirai M.Y. Hayashi H. Chino M. Naito S. Fujiwara T. Brunold C. Rennenberg H., De Kok L.J. Stulen I. Davidian J.-C. Sulfur Nutrition and Sulfur Assimilation in Higher Plants. P. Haupt Publ., Bern2000: 331-333Google Scholar). The association and dissociation of CSC in the absence or presence of OAS have been depicted in a preliminary reaction scheme in Fig.1.In this study we have applied SPR to elucidate the function of this protein complex. The homomeric subunits were found to behave similar to single polypeptide chains. This allowed modeling of interaction kinetics based on a simple A + B ⇔ AB principle and demonstrated that SPR-based BIAcore technology (21Chaiken I. Rose S. Karlsson R. Anal. Biochem. 1992; 201: 197-210Crossref PubMed Scopus (196) Google Scholar) can be applied to quantitative kinetic analysis of a multimeric protein complex. The assessment of OAS effects on CSC dissociation eliminates any function in substrate channeling common to other metabolic multimeric complexes. Instead, the kinetics obtained provide the first evidence for a function of the dissociation state of a metabolic enzyme complex as a regulatory switch for the entire upstream biosynthetic pathway.DISCUSSIONThis study provides a quantitative kinetic description of the effect of the putative effector OAS on the enzyme-enzyme interaction in the mitochondrial CSC from A. thaliana. As a prerequisite for a detailed kinetic investigation, a preparation protocol for the mitochondrial CSC from A. thaliana and its free subunits, SAT and OAS-TL, was established that allows rapid purification at high yield, purity, and activity after expression in E. coli. Within less than 2 h CSC representing up to 10% of total soluble bacterial protein was purified to apparent homogeneity. This corresponds to an ∼20-fold improvement of yield compared with previous preparations of recombinant CSCs (17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar, 19Wirtz M. Berkowitz O. Droux M. Hell R. Eur. J. Biochem. 2001; 268: 686-693Crossref PubMed Scopus (97) Google Scholar, 27Mino K. Yamanoue T. Sakiyama T. Eisaki N. Matsuyama A. Nakamishi K. Biosci. Biotechnol. Biochem. 1999; 63: 168-179Crossref PubMed Scopus (55) Google Scholar). In contrast to free cytosolic SAT from A. thaliana (17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar), the mitochondrial SAT analyzed in this study proved to be quite stable in the absence of OAS-TL. This difference may be intrinsic for the different compartmental SAT isoforms from A. thaliana or a result of the different fusion tags and isolation procedures.Our CSC preparation confirmed several characteristic features of the CSC that have been partly described for CSCs from bacteria (3Kredich N.M. Becker M.A. Tomkins G.M. J. Biol. Chem. 1969; 244: 2428-2439Abstract Full Text PDF PubMed Google Scholar, 5Cook P.F. Wedding R.T. Arch. Biochem. Biophys. 1977; 178: 293-302Crossref PubMed Scopus (42) Google Scholar, 27Mino K. Yamanoue T. Sakiyama T. Eisaki N. Matsuyama A. Nakamishi K. Biosci. Biotechnol. Biochem. 1999; 63: 168-179Crossref PubMed Scopus (55) Google Scholar) and plants (17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar, 28Droux M. Martin J. Sajus P. Douce R. Arch. Biochem. Biophys. 1992; 295: 379-390Crossref PubMed Scopus (84) Google Scholar) as follows: bound OAS-TL is much less active than the free enzyme; OAS can dissociate the CSC; OAS-TL released from the complex gains full activity as compared with free OAS-TL dimers. Based on these special properties an activity assay using OAS pretreatment of the complex with increasing OAS concentrations was developed that allowed us to correlate the activation of OAS-TL with the degree of dissociation of the CSC. The observed Michaelis-Menten constant for OAS of Km = 57 μm indicated a 50% dissociation already far below the millimolar OAS concentrations conventionally used to disrupt the complex in vitro (3Kredich N.M. Becker M.A. Tomkins G.M. J. Biol. Chem. 1969; 244: 2428-2439Abstract Full Text PDF PubMed Google Scholar, 17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar,19Wirtz M. Berkowitz O. Droux M. Hell R. Eur. J. Biochem. 2001; 268: 686-693Crossref PubMed Scopus (97) Google Scholar). Furthermore, significant shifts in the equilibrium between CSC and free subunits can be predicted in response to even small changes of OAS concentrations in vivo.A quantitative assessment of the kinetic behavior of SAT and OAS-TL interaction was required to gain evidence for a putative regulatory function of this metabolic protein complex system. However, dissociation kinetics of a hetero-oligomeric complex of 300–350 kDa might be expected to be quite complicated because of the multitude of possible interaction sites between protein subunits. This study shows that biomolecular interaction analysis using SPR can be suitable for the analytical description of such mechanisms. Binding characteristics and binding stability of real time interaction as detected by BIAcore were carefully monitored and evaluated with respect to feasibility of a simple A + B ⇔ AB model for association and dissociation of the CSC. In a first approximation, the binding kinetics observed indeed strongly suggest such tight SAT and OAS-TL homomers, respectively, that their interaction at the sensor surface can be sufficiently described with this model. Significant statistical deviation of the dissociation rate from an ideal mono-exponential function was only observed during long term monitoring (45 min). Intermediate steps during the dissociation reaction of the multimeric complex might be able to cause this difference or an overlay of complex dissociation with release of the SAT anchor protein from the nickel matrix of the chip. However, such bleeding of SAT was very minor (Fig. 4A). Whereas a SAT tetramer is assumed to bind two OAS-TL dimers (3Kredich N.M. Becker M.A. Tomkins G.M. J. Biol. Chem. 1969; 244: 2428-2439Abstract Full Text PDF PubMed Google Scholar, 28Droux M. Martin J. Sajus P. Douce R. Arch. Biochem. Biophys. 1992; 295: 379-390Crossref PubMed Scopus (84) Google Scholar, 30Warrilow A.G.S. Hawkesford M.J. J. Exp. Bot. 2000; 51: 985-993Crossref PubMed Scopus (61) Google Scholar), the loading of immobilized SAT with saturating concentrations of OAS-TL yielded a 2:1 ratio of SAT to OAS-TL. This may be caused by blocking of OAS-TL interaction domains on SAT subunits due to the fixed histidine-nickel-binding site, although such a steric inhibition was not observed during affinity tag isolations of the CSC (Fig. 2) (17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar,19Wirtz M. Berkowitz O. Droux M. Hell R. Eur. J. Biochem. 2001; 268: 686-693Crossref PubMed Scopus (97) Google Scholar). A 2:1 quaternary organization of the native complex cannot be excluded, because the SAT homomer from E. coli was recently reported (29Hindson V.J. Moody P.C.E. Rowe A.J. Shaw W.V. J. Biol. Chem. 2000; 275: 461-466Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) to consist of a dimer of trimers instead of a tetramer. It has to be considered, however, that these analyses were performed in the absence of OAS-TL.The equilibrium dissociation constant for the interaction of mitochondrial SAT and OAS-TL in the absence of any effector (KD = 25 ± 4 nm) is in agreement with the KD value of 41 nm determined for the interaction between cytosolic SAT from A. thalianaand plastid OAS-TL from spinach (17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar). The application of the SPR-based biosensor method with an immobilized ligand thus confirms the analysis of the heterologous CSC that had been determined with soluble components in a velocity versus [OAS-TL] plot (17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar). TheKD of the CSC indicates a very stable complex that in fact is comparable with the interactions of antibody-antigen and receptor-ligand partners.The effect of OAS on the dissociation of the CSC on the BIAcore chip surface was best described by a Hill plot that suggested strong cooperativity for the dissociation of SAT and OAS-TL. According to this fit, 77 μm OAS is sufficient to achieve the half-maximal dissociation rate. This is in exactly the same range as theKm value of 58 μm OAS required to adjust 50% equilibrium dissociation state of the complex. Changes of OAS concentrations in a very narrow range thus are bound to cause fast and almost complete association or dissociation of the CSC. It is important to note that the OAS concentrations relevant for complex dissociation are far below the substrate affinities of free and bound OAS-TL enzymes for OAS that range around 1 mm (9Jost R. Berkowitz O. Wirtz M. Smith L. Hawkesford M.J. Hell R. Gene (Amst.). 2000; 253: 237-247Crossref PubMed Scopus (99) Google Scholar, 28Droux M. Martin J. Sajus P. Douce R. Arch. Biochem. Biophys. 1992; 295: 379-390Crossref PubMed Scopus (84) Google Scholar, 30Warrilow A.G.S. Hawkesford M.J. J. Exp. Bot. 2000; 51: 985-993Crossref PubMed Scopus (61) Google Scholar), whereas dissociation constants of OAS-TL dimers for OAS are below 10 μm. 2M. Wirtz and R. Hell, manuscript in preparation.Dissociation effects can therefore become effective before substantial catalytic activity can take place. Taken together, such kinetic behavior provides an ideal molecular switch for regulation of downstream processes in a threshold-dependent manner.Indeed, a regulatory function for OAS in regulation of sulfur metabolism has been suspected (7Leustek T. Martin M.N. Bick J.-A. Davies J.P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 141-166Crossref PubMed Scopus (503) Google Scholar, 14Smith F.W. Hawkesford M.J. Ealing P.M. Clarkson D.T. Vanden Berg P.J. Belcher A.R. Warrilow A.G. Plant J. 1997; 12: 875-884Crossref PubMed Scopus (238) Google Scholar, 31Neuenschwander U. Suter M. Brunold C. Plant Physiol. 1991; 97: 253-258Crossref PubMed Scopus (84) Google Scholar, 32Hell R. Habilitation Thesis, Ruhr-Universität Bochum 1997. Shaker, Aachen, Germany1998: 23-41Google Scholar). OAS concentrations have been described to differ between 2 and 56 nmol/g FW, respectively, in rosette leaves of 9-week-old sulfur-sufficient and sulfur-limited A. thaliana plants (20Awazuhara M. Hirai M.Y. Hayashi H. Chino M. Naito S. Fujiwara T. Brunold C. Rennenberg H., De Kok L.J. Stulen I. Davidian J.-C. Sulfur Nutrition and Sulfur Assimilation in Higher Plants. P. Haupt Publ., Bern2000: 331-333Google Scholar). In sulfur-deprived potato leaves OAS levels increase from 3 to 6 nmol/g FW to 220 nmol/g FW within 8 days. 3L. Hopkins and M. J. Hawkesford, personal communication. The cellular contents of OAS increase from 0.3 to 3.8 nmol/g FW in A. thaliana suspension cultures within 72 h after transfer to sulfate-depleted medium. 4M. Wirtz and R. Hell, manuscript in preparation.Assuming that OAS is not localized to the vacuole, a calculation based on relative volumes of cell compartments (33Bowsher C.G. Tobin A.K. J. Exp. Bot. 2001; 52: 513-527Crossref PubMed Google Scholar) yields cellular OAS concentrations between 10 and 60 μm during sulfur-sufficient and 60–200 μm during sulfur-limited conditions. Intracellular OAS fluctuations would thus occur in the critical range of CSC dissociation.Our findings therefore provide for the first time the quantitative basis for essential elements of a hypothesis for the function of the CSC (17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar, 32Hell R. Habilitation Thesis, Ruhr-Universität Bochum 1997. Shaker, Aachen, Germany1998: 23-41Google Scholar). In this model SAT and OAS-TL are associated in the presence of sufficient sulfur in the cell, because sulfide stabilizes the complex and OAS levels are low (Fig.6). OAS leaves the CSC because of the low affinity of bound OAS-TL and is consumed to produce cysteine by free OAS-TL dimers (3Kredich N.M. Becker M.A. Tomkins G.M. J. Biol. Chem. 1969; 244: 2428-2439Abstract Full Text PDF PubMed Google Scholar, 17Droux M. Ruffet M.-L. Douce R. Job D. Eur. J. Biochem. 1998; 255: 235-245Crossref PubMed Scopus (203) Google Scholar). If the cells encounter sulfate deficiency, sulfide levels will drop and OAS will accumulate to concentrations that are sufficient to effectively dissociate the CSC. Consequently, SAT would become less active by degradation or modification, and at the same time OAS could trigger the de-repression sulfate uptake and assimilation (14Smith F.W. Hawkesford M.J. Ealing P.M. Clarkson D.T. Vanden Berg P.J. Belcher A.R. Warrilow A.G. Plant J. 1997; 12: 875-884Crossref PubMed Scopus (238) Google Scholar, 16Koprivova A. Suter M. den Camp R.O. Brunold C. Kopriva S. Plant Physiol. 2000; 122: 737-746Crossref PubMed Scopus (223) Google Scholar, 31Neuenschwander U. Suter M. Brunold C. Plant Physiol. 1991; 97: 253-258Crossref PubMed Scopus (84) Google Scholar). Thereby the system becomes reversible, because sulfate enters the cell and after reduction sulfide reacts with the accumulated OAS via free OAS-TL dimers. Lowered OAS levels now promote formation of the CSC from free OAS-TL and reactivated or newly synthesized SAT. It is concluded that the plant CSC functions as a molecular sensor system that monitors the sulfur status of the cell and controls sulfate assimilation and cysteine synthesis according to the availability of sulfate.FIG. 6Model of the function of OAS-driven reversible protein-protein interaction of SAT (squares) and OAS-TL (circles) in the regulation of cysteine synthesis rate and expression of sulfate uptake and assimilation genes. Active forms of the enzymes are represented by open symbols, and inactive or low affinity forms, respectively, are shown by dark symbols. Open arrowsindicate transitions of a cell between sulfur-sufficient and sulfur-limited conditions.View Large Image Figure ViewerDownload (PPT) The interaction of proteins is essential for the function of living cells (1Huang X. Holden H.M. Raushel F.M. Annu. Rev. Biochem. 2001; 70: 149-180Crossref PubMed Scopus (309) Google Scholar, 2Miles E.W. Rhee S. Davies D.R. J. Biol. Chem. 1999; 274: 12193-12196Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Monitoring of biomolecular interaction analysis can be achieved by BIAcore technology that is based on surface plasmon resonance (SPR).1 The physical phenomenon of SPR is widely used to visualize macromolecular interactions in real time with the advantages of no labeling requirements and the option to determine kinetic rate constants. It is mostly applied to quantification of antigen/antibody binding, receptor ligand screening, or characterization of protein modification. It has not been used so far to investigate enzyme-enzyme interactions, in particular with respect to regulation of the metabolic activity of multienzyme complexes. In this study we have applied SPR to analyze the interaction of a hetero-oligomeric metabolic protein complex using the enzymes of cysteine biosynthesis from mitochondria of Arabidopsis thaliana. Serine acetyltransferase (SAT; EC 2.3.1.30) generates the activated sulfide acceptor O-acetylserine (OAS) from serine and acetyl-CoA. O-Acetylserine (thiol)-lyase (OAS-TL; EC 4.2.99.8) then inserts free sulfide in a β-replacement reaction to synthesize cysteine and acetate. One tetramer of SAT and two dimers of OAS-TL are presumed to form the hetero-oligomeric cysteine synthase complex (CSC) that was first described for Salmonella typhimurium (3Kredich N.M. Becker M.A. Tomkins G.M. J. Biol. Chem. 1969; 244: 2428-2439Abstract Full Text PDF PubMed Google Scholar, 4Kredich N.M. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umberger E. Escherichia coli and Salmonella typhimurium. American Society for Microbiology, Washington, D. C.1996: 514-527Google Scholar). Protein-protein interactions of such metabolic enzymes mostly serve to channel substrate intermediates between active sites of sequential reaction steps of a pathway. The advantages of these quaternary organizations include efficient catalysis rates and stabilization of intermediates by prevention of losses of intermediates into solution by diffusion (1Huang X. Holden H.M. Raushel F.M. Annu. Rev. Biochem. 2001; 70: 149-180Crossref PubMed Scopus (309) Google Scholar, 2Miles E.W. Rhee S. Davies D.R. J. Biol. Chem. 1999; 274: 12193-12196Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). However, substrate channeling is not realized in the CSC, because a ready release of the reaction intermediate O-acetylserine (OAS) into the surrounding solution was observed for the bacterial complex (5Cook P.F. Wedding R.T. Arch. Biochem. Biophys. 1977; 178: 293-302Crossref PubMed Scopus (42) Google Scholar). The reason for the association of SAT and OAS-TL in the CSC is therefore unknown, despite the fact that the importance of this final step of sulfate assimilation is comparable with the fixation of ammonia by glutamine synthetase in nitrate assimilation (4Kredich N.M. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umberger E. Escherichia coli and Salmonella typhimurium. American Society for Microbiology, Washington, D. C.1996: 514-527Google Scholar, 6Saito K. Curr. Opin. Plant Biol. 2000; 3: 188-195Crossref PubMed Scopus (251) Google Scholar, 7Leustek T. Martin M.N. Bick J.-A. Davies J.P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 141-166Crossref PubMed Scopus (503) Google Scholar). In plants the synthesis of cysteine takes place in cytosol, plastids, and mitochondria. Nuclear encoded cDNAs of SAT and OAS-TL for each compartment-specific isoform have been cloned from several plants including A. thaliana (8Noji M. Inoue K. Kimura N. Gouda A. Saito K. J. Biol. Chem. 1998; 273: 32739-32745Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 9Jost R. Berkowitz O. Wirtz M. Smith L. Hawkesford M.J. Hell R. Gene (Amst.). 2000; 253: 237-247Crossref PubMed Scopus (99) Google Scholar). Expre
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