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1 Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006, pp 대장균의동역학네트워크모델을이용한 L-threonine 생합성에관한모사연구 om Çmsk ne e 1 (2006 1o 12p r, o 3p }ˆ) Simulation Study of Dynamic Network Model for L-Threonine Biosynthesis in Escherichia coli Uisub Jung and Jinwon Lee Department of Chemical and Biomolecular Engineering, Sogang University, 1, Sinsu-dong, Mapo-gu, Seoul , Korea (Received 12 January 2006; accepted 3 February 2006) k h l l q l L-threoninep l m p r r l l o l L-aspartate l L-threonine vp k o p pd m. L-threonine l m p r r p rp l pel rne. l o p L-aspartate 5 mm, ATP 5 mm, NADPH 2 mmp r r rp l v p p m. r r L-lysine, L-methionine, L-glutamate r r l v p ll. r r L-serine, L-cysteine L-threoninep n r rp l v p p ll. q l lv L-aspartatep L-threonine p ll, L-threoninep l r D,L-aspartic β-semialdehyde l m. Abstract In order to investigate the effect of inhibitors on L-threonine biosynthesis in Escherichia coli, we have constructed a metabolic network model of amino acid biosynthesis from L-aspartate to L-threonine by using available informations from literatures and databases. In the model, the effects of inhibitors on the biosynthesis of L-threonine was included as an appropriate mathematical form. For simulation study, we used initial values as L-aspartate 5 mm, ATP 5 mm, NADPH 2 mm, and observed the concentration changes of intermediate metabolites over concentration changes of respective inhibitors. As a result, we found that concentrations of intermediate metabolites were not significantly changed over concentration changes of L-lysine, L-methionine, and L-glutamate. But, there were considerable changes of intermediates over concentration changes of L-serine, L-cysteine, and L-threonine, which can be considered as essential effectors on L-threonine synthesis. Contrary, the synthesis of L-threonine seems to be not related to the amounts of L-aspartate, and inversely proportional to the accumulated amount of D,L-aspartic β-semialdehyde. Key words: L-Threonine Synthesis, Metabolic Modeling, Escherichia coli, in silico Simulation, Metabolic Inhibitors 1. l k tl rp TCA cycle vp L-oxaloacetate r ~ l lv k p L-threonine, L-lysine, L-methionine, L-isoleucinep p [1]. t L-threoninep ep CH 3 CH(OH)CH(NH 2 )COOHp mk To whom correspondence should be addressed. jinwonlee@sogang.ac.kr k tp p q l 2 p ˆ oq v 4 p p v~(l-threonine, D-threonine, L- allothreonine, D-allothreonine) pp }ll sq p L- threoninep. vp k p v vl e vl r. l o L-threoninep p v kv l p pn l n p k v p. L-threoninep k vp n k p [2]. 97

2 98 rp Ëpvo l L-aspartatel L-homoserinep L- glycinel threonine aldolase qn l. L-threonine p r p v p v, ov, ˆ (Elastin) l vp tn p. l ~ l l p k r pp L-threoninep e e e qk o kp q o, pp v m l rp k p k r p [1, 2]. pl l l p tn L-threoninep l o p l L-threonine l l r r p r l rneˆ q l p L- threonine p ƒ l (simulatuion) p pn l v p e l v p q p L-threoninep l r r t l r r m p vl k. r tp rp q v o l q p l lv q p k L-threoninep k v t L-aspartatel L-homoserinep rp ˆ m [3-5]. q l p L-threonine o l r l Žl l lv q m r o l r p BioCyc, KEGG, EMT project BRENDAp q ˆp l L-aspartate eqrp l L-threonine vp 5 p m [6-8]. L-threonine l l l p l m p s (cofactor), pm(metal ion), r r(inhibitor), r(activating compound) p s l L-threonine o l rne [9]. p rp q p L-threonine ~ p L- aspartatel β-aspartyl phosphate ~ (enzyme complex) qn l aspartokinase Im aspartokinase III qn p. p p k e (stoichiometry)p v m p r r p s l. Aspartokinase Ip nl s p L-threoninep r aspartokinase IIIp nl L-lysine L-glutamate r r l p L-threonine Šk } l Žl r~ k rp s tl lrp on n L-threoninep pn l Fig. 1 p L-aspartatel L-threonine vp o m [3-5]. l p v }k p r r Fig. 1l e m. L-threonine t r rp m p q p homoserine kinase(ec ) p r rl m p. p r r p p r v k p p r (L-serinep rn) L-threoninep vp l r r l p p ˆ L-threonine Šk i h l q p L-threonine o pn l k e(stoichiometry)p m (Table 1). L- aspartatel L-threoninep pp pp er qn p ~ l ~ qn aspartokinase I, IIIl p l p pp l p [7]. 5 p p t k pp k p(reversible reaction)p p v pp l p(irreversible reaction)p. l AK I/IIIm HSK l p phosphate ASADHm TS l. Table 1. Stoichiometric equations for L-threonine biosynthesis metabolic network Enzyme Stoichiometry AK I/III ASP + ATP ASPP + ADP ASADH ASPP + NADPH ASPSA + NADP + + P HDH i ASPSA + NAD(P)H HSER + NAD(P) + HSK HSER + ATP PHSER + ADP TS PHSER + H 2 O THR + P i Fig. 1. L-threonine biosynthesis metabolic pathway in E. coli: Inhibitors (SER, CYS, GLU, LYS, MET, THR). o44 o k

3 q p l o p pn L-threonine l l i l l m q p L-threonine l o (simulation) o p p v/ p(asadh prn: v/ p)p p. p k ep A+B Ë P+Q, Pm Q vp Am Bl qrp p. v p p ol v p l r r l p p f p l m p. p v p ep p r ˆ rp (1)e p ˆ p [10, 11]. v K P V f AB PQ K eq = P Q K A A K B B K Q l, V f r pp (V max ) ˆ. op (1)ep l pp r V r p Haldane relationshipp Ž p pn (2)e p l o l. (1) V f K P K Q = V r K A K B L-threonine 6 p m l pel m p r r p o q s m. p sp l r rm o q l }p r r tl L-threonine o m q pr p v p v p v p rp s m. r r p p pl qr(competitive)p p v qr(noncompetitive)p r v s m. l L-threonine l 6 p r r(l-serine, L-cysteine, L-threonine, L-lysine, L-methionine, L-glutamate) }k p p rp l pel r r m pq e s r pep m. p r r p l r v kp (L-serinep n rn) p p l m p p p m (L-threoninep n p l m p ). L-threonine p pe p rp p Table 2l ˆ l [3, 6-9]. (2) Table 2. Enzyme kinetics for L-threonine biosynthesis in E. coli metabolic network Enzyme Kinetics V [ ASPP] [ ADP] AKl [ ASP] [ ATP] [ THR] AK I K ithr h THR K ASP [ ASPP] K ASP [ ASP] K [ ADP] [ THR] ATP [ ATP] K α K ithr h THR ASPP K ADP AK III V [ ASPP] [ ADP] AK3 [ ASP] [ ATP] [ LYS] h LYS [ GLU] K [ ASPP] [ K ilys ] K ASP [ ASP] K [ ADP] iglu K ASPP ATP [ ATP] K ADP ASADH HDH HSK TS V [ ASPSA] [ NADP] [ P ASD [ ASPP] [ NADPH] i ] [ ASPSA] K ASPP [ P i ] [ ASPP] K [ NADP] K ASPSA K Pi NADPH [ NADPH] K NADP V [ HSER] [ NADP] HDH [ ASA] [ NADPH] [ THR] K ithr h THR [ CYS] [ SER] K [ HSER] [ THR] K α K ithr h THR icys K ASA [ ASA] K [ KADP] iser K HSER NADPH [ NADPH] K NADP V HK [ HSER] [ ATP] [ ATP] K HSER [ THR] [ LYS] [ MET] [ CYS] [ GLU] HSER [ ] KATP [ HSER] [ ATP] K iatp K ithr K ilys K imet K CYS K iglu K ihser V TS [ PHSER] [ PHSER] K PHSER Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

4 100 rp Ëpvo 2-4. n o L-threonine rl ASP ASPP l ~ l m p AKÏ THRp r r l p p k r pp p THRp ASPl k d (allosteric) qr(competitive)p qn [12, 13]. ~ l qn p AK IIIp nl L-lysinep qr(noncompetitive)p, m Ž (cooperative) r L- glutamate le r r v qrp p l m p [14-16]. p ASADH L-threonine m n v r r l p vp l p s l. HDH r v s p L-serine, L-cysteine L-threoninep pp L-threoninep nl qr(non-competitive) r r m p [18-20]. HSK s lv r r t L-serinep rn r rp m p p s l [21-24]. vp L-homoserinep n 1 mm p p, vp ATP 3 mm p p v r r l p p k r p. THRp L-homoserine l qr(competitive)p k d (non-allosteric) r rp, L-lysinep nl L-homoserine qrp(noncompetitive) r r k r p [3]. L-glutamate, L- methionine, L-cysteinep n HSK p r r l p [9]. L-threonine rl v p TS pl ASADH m v vrrp r r l p vp l p s l (simulation) 3. y l l q l L-threonine o l p op, p pe Ž p o l r q m. p m Ž p Table 3l ˆ l [3, 9]. n (simulation) p ~ o pn COPASI p n m [11] s lp Ž COPASIp time course simulationp pn l L-aspartatep 5mMp q p vp β-aspartyl phosphate, D,L-aspartic β-semialdehyde, L-homoserine, O-phosphohomoserine L-threoninep l v }p 10 kp 1 p l 5 p 100 kp p ll, p p m. p o v p p L-aspartate 5 mm, ATP 5 mm, NADPH 2 mm r v v p p 0 mm, L-threonine l r r qn v p r p 1 mm r m., L-threoninep r rp s p l r r p m l (simulation) p o p 0 mm r L-threoninel p r m p l r r 1 mm r L-threoninep p 0 mm, 0.01 mm, 0.1 mm, 1 mm r l m. Table 3. Parameters of enzyme kinetics for L-threonine biosynthesis in E. coli Enzyme K m (mm) Inhibition AK ASP 0.97±0.48 K ithr 0.167±0.003 mm ATP 0.98±0.5 h THR 4.09±0.26 ASPP 0.017±0.004 a 2.47±0.17 ADP AK ASP 0.32±0.08 K ilys 0.391±0.08 mm ATP 0.22±0.02 h LYS 2.8±1.4 ASPP 0.017±0.004 K iglu 128 mm ADP 0.25 ASADH ASPP 0.022± NADPH 0.029±0.002 ASA 0.11±0.008 NADP ±0.02 P i 10.2±1.4 HDH ASA 0.24±0.03 K ithr mm M 1 NADPH 0.037±0.006 h 1.41 HSER 3.39±0.006 a 3.93 NADP ±0.006 K icys 0.1 mm o44 o k K iser 0.1 mm HSK HSER 0.11 K ithr 1.09 mm ATP K ilys 9.45 mm TS PHSER 0.31±0.03 K ihser K iatp K imet K icys K iglu 4.7 mm 4.35 mm 35 mm 1 mm 0.5 mm

5 q p l o p pn L-threonine l l 101 Fig. 2. Intermediate metabolite concentration profiles in the presence of L-serine as an inhibitor n o glutamate, lysine, methioninei i l l s L-threonine rl m p r r t L-glutamate, L-lysine, L-methioninep nl p r r 0.01 mm, 0.1 mm, 1 mm, 10 mm tl (simulation) v p e p k. ASP, ASPP, ASA, HSER, PHSER, THRp p r rp v Ž p p llp, v p Fig. 2m d Ž p m. ASPp nl 100 p v k 3.33 mmr pr Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

6 102 rp Ëpvo mp, ASPP (simulation) 1 l p k mmp ˆ m. ASA 100 k v 100 l k 1.42 mm r p ov HSER PHSERp 6 l 9 p, 9 l 12 pl ˆ m. v p THRp 100 p v nl k 0.23 mm r ov p p n o serine i i L-serinep r(glycolysis)p v vp L-pyruvate k p L-threoninep t p L-homoserine p r k p. pl L-serine p q p L-threonine rl homoserine dehydrogenase (HDH, EC ) l n tn r r l p p k r p [17]. L-serinep r p m php m p n. L-serinep 100l 200 mmp ph 7.5p n D,L-aspartic β-semialdehydem L-homoserine pl qn HDH p r p l p r r l p [18]. l l r rp L-serinep t po r r 1 mm r(l-threoninep n p 0 mmp r) L-serinep e L-threonine p e 100 k (simulation) r rp 0.01 mm, 0.1 mm, 1 mm, 10 mmp eˆ n v p k Fig. 2m p p ˆ p p m. ASP p n r rp L-serine v ASPp v p p l (10 mmp nl k 1.8 mm r ). ASPPp p 1 l p e p v tl l 100 p v p s l k 0.03 mm r p ASPP q l sq. ASA p nl L-serinep v ASAp p r l (10 mmp nl k 1.7 mmr r), HSERp n (simulation) k 4 l p m 15 l tl p p plp (0.01 mmp nl k mmr ) 100 l p 0 mml sq. L-serinep p m v p k pl. PHSERp nl HSERp m d p p k 7 m 20 l PHSERp tl l. v p e THR p Sq p L-serinep rp p THR q l sq p p l (10 mmp nl k 0.06 mmr sq) n o cysteine i i L-cysteinep n q l L-serinep lv k p homoserine dehydrogenase(hdh, EC )m homoserine kinase(hsk, EC ) l m p r r k r p. L-cysteinep HSK p n vp ATPl qrp r rp vp L-homoserinel q rp r r qnp [20, 21]. L-serine v r rp reˆ L-cysteine o44 o k p tl (simulation) p COPASI p n l 100 kp q v p Fig. 3 p ll. L-serine v r r L-cysteinep 0.01 mm, 0.1 mm, 1 mm, 10 mm tlp r~rp p d m. l rp HSERp L-serinep n p k 1/2r tl p p plp L-cysteinep 10 mmp nl k 15 q l sq HSERp s (0.01 mm, 0.1 mm, 1 mm) p p p ˆ. PHSERp n L-cysteinp 0.01 mm 0.1 mmp L-serinep n k j p p p l. p p L-threonine p l L-serine l p ll n o L-threoninei i l l v p L-threonine le s p el r r aspartokinase I(AKÏ, EC ), homoserine dehydrogenase(hdh, EC )m homoserine kinase(hsk, EC ) l m p k r p [19, 21-23]. AK Ip nl vp L-aspartatem k d (allosteric) qr (competitive)p r HDH l L-threonine p k d (allosteric) qn qr(non-competitive) p p r. v p HSK l L- homoserinem qr(competitive)pv k d (non-allosteric) m p. r r p l m p l Fig. 4 m p r r p m l Ž p m. ASP p L-threoninep 1 mmp rn e d ˆ ASPP L-threonine p 0l 0.1 mm k 1 p v p mm v m 1mMp mm. ASA (simulation) 70 r v e r 1 mmp n p r 70 kv p m. HSERp r r l Ž p d L-serinel 7.6, L-cysteinep nl 4 r mmp ˆ l. PHSERp nl HSER d Ž p p L-serinel 4.6, L-cysteinep nl 5 r mmp ˆ l. THRp v m s p p t l p p pl. 4. l l ƒ l (simulation) p pn l p l rp p o r p e m, p o tp q (Escherichia coli) l L- threonine p (simulation) p f er q n L-threoninep e q m. l p o l sl p q p o erm n p o L-threonine l l p r r s p l l

7 q p l o p pn L-threonine l l 103 Fig. 3. Intermediate metabolite concentration profiles in the presence of L-cysteine as an inhibitor. rn m. l 14 p v(r r ), 5 p p(3 p l p 2 p l p)p L-threonine pe p m. (simulation) l p o l q oq n l pe Ž l l m. p p pn l L-threonine (simulation) o p L-aspartate 5 mm, ATP 5 mm, NADP 2 mm r r r 1 mm r m. p r rl m p p m., q l r D,L-aspartic β-semialdehyde l s p L-threonine l m p p p m. m, r r L-serinep nl L-serinep Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

8 104 rp Ëpvo Fig. 4. Intermediate metabolite concentration profiles in the presence of L-threonine as an inhibitor mm, 0.1 mm, 1 mm, 10 mmp v 100 q l r D,L-aspartic β-semialdehyde 0.8 mm, 1.0 mm, 1.4 mm, 1.7 mmpmp s p L-threoninep 0.55 mm, 0.45 mm, 0.23mM, 0.07 mmpl. v L-threoninep l r D,L-aspartic β-semialdehydel m. o44 o k r r L-serine, L-cysteine L-threoninep np r r p (simulation) m p p Žk l. L-serine L-cysteinp nl d Ž p mp rp L-homoserinep p r r L-serinep L-cysteinep n 1/2 p l ( r rp p 0.01 mmp L-serine

9 q p l o p pn L-threonine l l 105 p n mm, L-cysteinep n 0.06 mm). L-threonine p nl r r 0 mm, 0.01 mm, 0.1 mmp m 1 mm p v p p Ž p m. l L-threonine r r p s l v p e m L-threonine l m p r r p kk lv, p q p r~r p o mp, L-aspartate eqrp (simulation) l p erm n l p l n p m o l l p l lp q pn l r p Ž p l n rp q p edšp pp p. p edš l l(m n )p vol p l l lp pl. k AK I/II : aspartate kinase (EC ) ASADH : aspartate-semialdehyde dehydrogenase (EC ) HDH : homoserine dehydrogenase (EC ) HSK : homoserine kinase (EC ) TS : threonine synthase (EC ) ASP : L-aspartate ASPP : β-aspartyl phosphate ASA : D,L-aspartic β-semialdehyde HSER : L-homoserine PHSER : O-phospho-homoserine THR : L-threonine GLU : L-glutamate LYS : L-lysine MET : L-methionine SER : L-serine CYS : L-cysteine ATP : adenosine 5-triphosphate ADP : adenosine 5-diphosphate NADPH : dihydrotriphosphopyridine nucleotide y 1. Stephanopoulos, G. N., Aristidou, A. A. and Nielsen, J., Metabolic Engineering: Principles and Methodologies, Academic press (1998). 2. Faurie, R., Kimura, E., Marz, A., Mockel, B., Mueller, U., Pfefferle, W. and Thommel, J., Microbial Production of L-Amino Acids, Springer Verlag(2003). 3. Chassagnole, C., Raïs, B., Quentin, E., Fell, D. A. and Mazat, J. P., An Integrated Study of Threonine-pathway Enzyme Kinetics in Escherichia coli, Biochem. J., 356(2), (2001). 4. Raïs, B., Chassagnole, C., Letellier, T., Fell, D. A. and Mazat, J. P., Threonine Synthesis from Aspartate in Escherichia coli Cell-free Extracts: Pathway Dynamics, Biochem. J., 356(2), (2001). 5. Chassagnole, C., Fell, D. A., Raïs, B., Kudla, B. and Mazat, J. P., Control of the Threonine-synthesis Pathway in Escherichia coli: a Theoretical and Experimental Approach, Biochem. J., 356(2), (2001) Segel, I. H., Enzyme Kinetics: Behaviour and Analysis of Rapid Equilibrium and Steady-State, Wiley(1975) Starnes, W. L., Munk, P., Maul, S. B., Cunningham, G. N., Cox, D. J. and Shive, W., Threonine-Sensitive Aspartokinase-homoserine Dehydrogenase Complex, Amino Acid Composition, Molecular Weight, and Subunit Composition of the Complex, Biochemistry, 11(5), (1972). 13. Veron, M., Falcoz-Kelly, F. and Cohen, G. N., The Threoninesensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K12. The Two Catalytic Activities are Carried by two Independent Regions of the Polypeptide Chain, Eur. J. Biochem., 28(4), (1972). 14. Keng, Y. F., Viola, R. E., Specificity of Aspartokinase III from Escherichia coli and an Examination of Important Catalytic Residues, Arch. Biochem. Biophys., 335(1), 73-81(1996). 15. Truffa-Bachi, P., Microbial Aspartokinases; The Enzymes, 3rd Ed. (Boyer, P.D., ed.), Academic Press, 8, (1973). 16. Funkhouser, J. D., Abraham, A., Smith, V. A. and Smith, W. G., Kinetic and Molecular Properties of Lysine-sensitive Aspartokinase. Factors Influencing the Lysine-mediated Association Reaction and Their Relationship to the Cooperativity of Lysine Inhibition, J. Biol. Chem., 249(17), (1974). 17. Hama, H., Kayahara, T., Tsuda, M., Tsuchiya, T., Inhibition of Homoserine Dehydrogenase I by L-serine in Escherichia coli, J. Biochem., 109(4), (1991). 18. Wedler, F. C. and Ley, B. W., Kinetic and Regulatory Mechanisms for (Escherichia coli) Homoserine Dehydrogenase-I. Equilibrium Isotope Exchange Kinetics, J. Biol. Chem., 268(7), (1993). 19. James, C. L. and Viola, R. E., Production and Characterization of Bifunctional Enzymes. Domain Swapping to Produce New Bifunctional Enzymes in the Aspartate Pathway, Biochemistry, 41(11), (2002). 20. Burr, B., Walker, J., Truffa-Bachi, P. and Cohen, G. N., Homoserine Kinase from Escherichia coli K12, Eur. J. Biochem., 62(3), (1976). 21. Huo, X. and Viola, R. E., Substrate Specificity and Identification of Functional Groups of Homoserine Kinase from Escherichia coli, Biochemistry, 35(50), (1996). 22. Huo, X. and Viola, R. E., Functional Group Characterization of Homoserine Kinase from Escherichia coli, Arch. Biochem. Biophys., 330(2), (1996). 23. Theze, J., Kleidman, L., St. Girons, I., Homoserine Kinase from Escherichia coli K-12: Properties, Inhibition by L-threonine, and Regulation of Biosynthesis, J. Bacteriol., 118(2), (1974). Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

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<30372E31362D323028BDC5C7F6C5C32DB9CCB1B970626D292E666D> k m v r1 r1 16~20 (2009) J. Korean Academy of Managed Care Pharmacy Vol. 1, No. 1 Pharmacy Benefit Management iç 1# x sƒ, 1 w w w Overview on Pharmacy Benefit Management in USA Kyung Eob ChoiGand Hyun