Báo cáo khoa học: Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein doc

Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein Shuqing Chen 1, *, Sarah Vincent 2 , David B. Wilson 1 and Bruce Ganem 2 1 Department of Molecular Biology and Genetics and 2 Department of Chemistry and Chemical Biology, Cornell University, NY, USA The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the L -tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to charac- terize the CM, PDH and regulatory domains. Residues 1–88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94–373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooper- ative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain. Keywords: chorismate mutase; E. coli T-protein; prephenate dehydrogenase. The final step in the biosynthesis of tyrosine in Escherichia coli and other enteric bacteria is the transamination of p-hydroxyphenylpyruvate, which is produced in two sequential chemical reactions from chorismic acid in nature’s shikimic acid metabolic pathway [1,2]. In the first reaction, chorismate undergoes a Claisen rearrangement to form prephenate, which is catalyzed by chorismate mutase (CM; EC 5.4.99.5). In the second reaction, prephenate undergoes NAD + -mediated oxidative decarboxylation to p-hydroxyphenylpyruvate, which is catalyzed by prephenate dehydrogenase (PDH; EC 1.3.1.12). In E. coli, both the CM and PDH activities are located in a single, bifunctional protein known as the T-protein, which is encoded by the tyrA gene. Tyrosine (Tyr) is an end product inhibitor of both CM and PDH, and induces aggregation of the T-protein [3]. An analogous bifunctional protein in E. coli, known as the P-protein, contains CM and prephenate dehydratase (PDT), and catalyzes the transformation of chorismate into phenylpyruvate in the biosynthetic pathway to phenylalanine. Domain mapping studies on the P-protein (386 amino acids, homodimer, molecular mass 43 kDa) have estab- lished that the CM, PDT, and regulatory activities reside in discrete, separable domains that can be subcloned and expressed [4–7]. The structure of the P-protein CM domain (residues 1–109), which has been solved by X-ray crystallography, reveals the key structural motif respon- sible for noncovalent dimer formation in the wild-type protein. However, biochemical studies aimed at mapping the various functional domains in the T-protein suggest a more complex spatial relationship of the catalytic sites. Primary sequence alignments between the T- and P-proteins indicate that CM in the T-protein is also located at the N-terminus, although the sequences share only approximately 25% similarity. Mutagenesis studies on the T-protein and kinetic studies using substrate analogs suggested that the CM and PDH reactions occurred at overlapping [8] or perhaps closely proximal [9] active sites. Strong evidence for two separate CM and PDH active sites comes from pH rate profile analyses [10] and from various substrate and product-based inhibitors that affect the two catalytic activities with differing degrees of selectivity [11]. At one extreme, a widely studied oxabicyclic mutase inhibitor has been shown to inhibit CM activity in the T-protein without affecting PDH activity [9]. More recently, a tricyclic diacid was reported to inhibit PDH activity in the T-protein without affecting CM activity [12]. The main objectives of this study were to investigate the various domain substructures, interactions, and allos- teric effects in the E. coli T-protein by genetically engineering and expressing fragments of tyrA.Using these techniques, we hoped to determine whether the CM and PDH activities could be separated into discrete, properly folded entities displaying good catalytic activity. We also hoped to ascertain whether a separate regulatory domain existed within the T-protein that was responsible for Tyr-induced end-product inhibition and T-protein aggregation. Finally, we hoped to gain an understanding Correspondence to B. Ganem, Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301 USA. Fax: + 1 607 255 6318, Tel.: + 1 607 255 7360, E-mail: bg18@cornell.edu Abbreviations: CM, chorismate mutase; PDT, prephenate dehydra- tase; PDH, prephenate dehydrogenase; WT, wild-type. Enzymes: chorismate mutase (EC 5.4.99.5); prephenate dehydrogenase (EC 1.3.1.12). *Present address: College of Pharmaceutical Science, Zhejiang University, Hangzhou 310031, P.R. China. (Received 25 October 2002, revised 11 December 2002, accepted 19 December 2002) Eur. J. Biochem. 270, 757–763 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03438.x of the detailed molecular interactions involved in T-protein dimerization. Experimental procedures Materials Unless indicated otherwise, all chemicals and biochemicals were purchased from Sigma, and enzymes were purchased from New England Biolabs. Strain E. coli BL21 Gold (DE3) competent cells (Stratagene) were used as the host for cloning, plasmid preparation and protein expression. Recombinant DNA method The tyrA gene, which codes for the T-protein, was subcloned from plasmid pKB45, a derivative of pMB9 that contains a 6-kb segment of E. coli chromosomal DNA [13]. Several primers (Table 1) were used to amplify specific fragments from pKB45. NdeIandXhoI sites were introduced into the primers at the N- and C-terminal coding sites, respectively, of the target fragments. A His tag was attached to the C-terminus of the wild-type (WT) T-protein as a means of simplifying the previously reported isolation [14] and purification [15] procedures. C-terminal His-tags were also attached to each fragment to facilitate subsequent purification. In order to promote the fidelity of PCR, GC-rich PCR kits were employed in amplification. DNA sequencing (Cornell BioResource Center) was carried out on every new plasmid to confirm that no mutations had been introduced by PCR. Novagen pET26b+ was used as the vector for all cloning. It has a kanamycin-resistant gene to facilitate screening for transformants. Expression All strains harboring plasmids were grown in LB (Luria– Bertani) medium or on LB plates containing kanamycin (60 lgÆmL )1 ). All strains were grown in LB containing kanamycin (60 lgÆmL )1 )at37°C for seed cultures and in LB without antibiotics inoculated 1 : 50 for large-scale enzyme production. Isolation and purification of the T-protein and cloned fragments thereof After induction with 1 m M isopropyl b- D -thiogalactoside at D 660 ¼ 0.8 and growth at 30 °C for 2.5 h, cells were collected by centrifugation at 10 000 g at 4 °C for 25 min. Cell pellets were resuspended in cold binding buffer (5 m M imidazole, 0.5 M NaCl, 20 m M Tris/HCl, pH 7.9), and the cells were ruptured at 2000 p.s.i. using a French press. Purification of the intact, His-tagged T-protein and of its cloned fragments was performed on His-tag resin (Novagen) following the manufacturer’s protocol. Peptide 1–88, without a His-tag, was obtained by mutating residue 89 to create a stop codon. The expressed peptide was purified by Q-Sepharose and Ultragel ACA54 column chromatography. Proteolytic digestion The purified T-protein was partly digested with papain by varying the time and quantity of papain. T-protein (20 lg) was dissolved in 100 lLof0.1 M NH 4 Ac, 0.004 M EDTA, 0.01 M cysteine (pH 6.8) and 0.4 lLof 0.1 mgÆmL )1 (1 : 50 ratio) or 2 lLof0.01mgÆmL )1 (1 : 1000 ratio) of papain were added. The reaction was incubated at 37 °Cand10lL samples were removed into tubes containing SDS gel loading buffer and put into a boiling water bath for 3 min at 0, 15, 30, 45, 60, 90, and 120 min intervals. All samples were then run on SDS/ PAGE gels. Enzyme assays Chorismate mutase and prephenate dehydrogenase activity assays were performed according to Davidson et al.[14] with 1 m M chorismate or 0.2 m M prephenate and 2 m M NAD, respectively. One unit of enzyme was defined as the amount of enzyme required to produce 1 lmol of product per minute at 37 °C. Specific activity was expressed as units per mg of protein. Kinetic studies Enzyme assays of the T-protein and derived fragments in the presence of Tyr were run at effector concentrations from 0to0.3m M , with substrate concentrations ranging from 0 to 1 m M or 2 m M based on the K m value to be measured. Controls were run for every assay. Values for the maximal Table 1. Primers used to clone T-protein peptides. Primer Sequence T01 5¢-GGT AGA CTC GAG TCA GTG GTG GTG GTG GTG GTG CTG GCG ATT GTC ATT CGC CTG ACG C-3¢ T02 5¢-GCT TAA GAG GTT TCA TAT GGT TGC TGA ATT G-3¢ PDH96 5¢-GGA TTT AAA ACA CAT ATG CCG TCA CTG CGT CCG GTG-3¢ PDH93 5¢-CGA CAA AGG ACA TAT GCA ACT TTG TCC GTC ACT GCG-3¢ PDH101 5¢-CCG TCA CTG CAT ATG GTG GTT ATC GTC GGC G-3¢ PDH93-336 5¢-CCA GTG CTC CAC CTC GAG TCA GTG GTG GTG GTG GTG GTG CTT ATC GCC CTG CTC CAG CAA-3¢ PDH93-316 5¢-CAA CTC AAT CGC CTC GAG TCA GTG GTG GTG GTG GTG GTG GAT TAA CGC CAG ATT ACG CTC TG-3¢ PDH93-296 5¢-GCT CTG ACG ACA TAA TCT CGA GTC AGT GGT GGT GGT GGT GGT GAG CCA ACA GTC GCC CGA CC-3¢ PDH93-276 5¢-CAT CGC CAG CTC AAG CTC GAG TCA GTG GTG GTG GTG GTG GTG AAG TTG CTC AAG CTG AAC AT-3¢ CM1-94 5¢-GCC ACC GCC GAC CTC GAG TCA GTG GTG GTG GTG GTG GTG AAG TGT TTT AAA TCC TTT GTC-3¢ CM1-108 5¢-CGA GAG GGT CAG CTC GAG TCA GTG GTG GTG GTG GTG GTG ACC GCC ACC GCC GAC GAT-3¢ 758 S. Chen et al.(Eur. J. Biochem. 270) Ó FEBS 2003 velocity (V max ) and the Michaelis constant (K m )were determined using standard rate equations in conjunction with the curve fitting options in the KALEIDAGRAPH program (Abelbeck Software). N-terminal analysis Samples of the proteolytic bands were prepared for N-terminal sequencing by electroblotting from the SDS gels after electrophoresis. An Immobilen-P membrane was prewet in methanol, and electrotransfer was performed following the manufacturer’s procedure (50 V, 1 h). Membranes were stained with 0.1% Commassie bright blue for 10 min and destained in 90% methanol, 7% acetic acid to a clear background. The band was cut out and N-terminal sequencing was performed on a PE/ Applied Biosystems Procise 492 by the Cornell Bio- Resource Center. Molecular mass estimation Molecular masses were determined by SDS gel electro- phoresis under denatured conditions and gel exclusion HPLC for determination of native molecular masses. Standard molecular mass markers (Invitrogen BenchMark Prestained Protein Ladder) were run on 12% or 17% SDS/ PAGE gels. A 600E Waters HPLC was used with a Pak Glass 300SW 8 · 300 mm column and 50 m M Tris/ HCl, pH 8.0, 50 m M NaCl buffer at a flow rate of 0.75 mLÆmin )1 . A Bio-Rad gel filtration standard was used to prepare a standard curve. Chemical cross-linking The C-terminal His-tagged T-protein was chemically cross- linked by a modified procedure as follows: 0.05 mg of T-protein was dissolved in 20 mL of 50 m M KH 2 PO 4 / K 2 HPO 4 buffer (pH 6.0), and 50% glutaraldehyde (0.83 mL) was added to give a final concentration of 2%. The reaction was run at room temperature for 22 h, then 0.5 mL of freshly prepared 2 M NaBH 4 /0.1 M NaOH was added to quench the reaction. After standing at room temperature for 20 min, 20 lL of 10% sodium deoxycho- late in 0.1 M NaOH was added followed by 0.5 mL of 100% trichloroacetic acid (w/v) and the mixture was incubated until the deoxycholate and protein precipitated. The sam- ples were centrifuged at 20 000 g for 20 min and the pellets were immediately dissolved in SDS/PAGE loading buffer containing dithiothreitol, boiled for 3 min and analyzed by electrophoresis on SDS/PAGE, using 17% acrylamide gels for proteins having molecular mass < 20 kDa and 12% acrylamide gels for proteins having molecular mass > 20 kDa. Results Expression Expression levels for all fragments lacking the native N-terminal sequence (plasmids PSQC2,3,4,5,6,7,8,13) were low. Good levels of expression were observed with all other fragments. By working at lower temperature (30 °C), the formation of inclusion bodies was suppressed, and expressed fragments were isolated from the soluble fractions. Activity of wild-type T-protein In assays of the WT T-protein, the specific activity for CM was 130 unitsÆmg protein )1 ,andthatforPDHwas 98 unitsÆmg protein )1 (Table 3). Both values were in good agreement with those determined by Davidson et al.[13]. However, prolonged storage of purified His-tagged T-protein at )80 °C, whether in storage buffer (0.1 M sodium citrate : 10% glycerol : 1 m M dithiothreitol, pH 7.5) or in assay buffer (0.1 M Mes, 0.051 M N-ethylmorpholine, 0.01 M diethanolamine, 1 m M EDTA, 1 m M dithiothreitol, 10% glycerol, pH 7.5) resulted in the loss of virtually all PDH activity (Fig. 1). Activity losses were somewhat smaller when protein was stored in the assay buffer. Because of the instability of the T-protein, all assays were performed on fresh enzyme. Controls indicated negligible loss of activity on the day that assays were conducted. The specific activity values reported in Table 3 were relative to freshly prepared enzyme (100% activity), and represented the highest values determined from the initial assays. Proteolysis studies When papain was used to digest the T-protein under limiting conditions (papain : T-protein ¼ 1 : 1000), a con- sistent pattern of fragments was detected having molecular mass values centered around 30 kDa and 10 kDa (Fig. 2). The N-terminal sequence of the 30 kDa fragment was determined to be TLCPSLRPVVIV, which corresponded to residues 93–104 of the T-protein. Essentially identical results were obtained when the T-protein was digested in the presence of Tyr (300 l M ), but without NAD + . Digestions carried out in the presence of higher con- centrations of papain (papain : T-protein ¼ 1:50) for limited periods of time revealed that the 30 kDa fragment Fig. 1. CM and PDH activity lost during storage. Ó FEBS 2003 E. coli T-protein catalytic and regulatory domains (Eur. J. Biochem. 270) 759 disappeared almost completely within 30 min, while the 10 kDa fragment was still detectable after 60 min (Fig. 3). Activity of cloned T-protein fragments Guided by the proteolysis results and using appropriately selected primer pairs, 14 new plasmids (Table 2) were constructed and used to express T-protein fragments corresponding to various regions of the T-protein sequence. The expressed proteins were designated with abbreviations indicating their T-protein origin and inclu- sive residues. The specific activities of both CM and PDH were determined for all engineered T-protein fragments (Table 3). The data indicate that all peptides containing the N-terminal 88 residues of the T-protein (entries 9–15) exhibited CM activity. However, the specific activity of all CM-active T-protein fragments was low. Even the largest such fragment, T/1–336, exhibited only approximately 5% of the native T-protein’s activity. The Michaelis constant, K m for CM activity in T/1–88 and T/1–336 were 1.7 ± 0.1 m M and 2.4 ± 0.5 m M , respectively. By com- parison, K m for the T-protein was 0.23 m M . None of the fragments exhibiting CM activity displayed PDH activity. T-protein fragments T/93–373 and T/96–373 (Table 3, entries 2 and 3) retain 25–50% of the PDH activity of the T-protein, but are devoid of CM activity. Fragment T/101–373 lacked PDH activity suggesting that residues 97–100 of the T-protein were essential for it (Table 3). Several additional T-protein fragments were studied (Table 3, entries 4–8) to refine the site of PDH activity. Fragments T/101–373, T/93–277, T/93–297, T/93–316, and T/93–336 displayed neither CM activity nor PDH activity. Expression levels of the truncated proteins in Table 3 entries 2–8 were significantly lower than for proteins in entries 9–15, which retained the native N-terminus. Feedback inhibition by Tyr In the absence of NAD + , the CM activity of fragments T/1–88, T/1–94 and T/1–108 was unaffected by Tyr at concentrations up to 300 l M . The CM activity of fragment Table 2. Primer pairs used in constructing plasmids for cloning T-protein fragments. Plasmid Primer T-protein fragment PSQC1 T02, T01 T/1–373 (T-protein) PSQC2 PDH93, T01 T/93–373 PSQC3 PDH96, T01 T/96–373 PSQC4 PDH101, T01 T/101–373 PSQC5 PDH93, PDH93-276 T/93–277 PSQC6 PDH93, PDH93-296 T/93–297 PSQC7 PDH93, PDH93-316 T/93–316 PSQC8 PDH93, PDH93-336 T/93–336 pSQC24 T/1–88 pSQC9 T02, CM1-94 T/1–94 pSQC10 T02, CM1-108 T/1–108 pSQC11 T02, PDH93-276 T/1–276 pSQC12 T02, PDH93-296 T/1–296 pSQC13 T02, PDH93-316 T/1–316 pSQC14 T02, PDH93-336 T/1–336 Fig. 2. Proteolytic digestion of the T-protein by papain at a ratio of 1 : 1000 (w/w). Lane 1, molecular mass standards; lane 2, 0 min; lane 3, 15 min; lane 4, 30 min; lane 5, 45 min; lane 6, 60 min; lane 7, 90 min; lane 8, 120 min. Fig. 3. The proteolytic digestion of T-protein by papain at a ratio of papain/T-protein ¼ 1:50(w/w).Lane 1, 0 min (enzyme added; some digestion observed); lane 2, 30 min; lane 3, 60 min; lane 4, molecular mass ladder. Table 3. CM and PDH activities of cloned segments of the T-protein. Enzyme activity (UÆmg )1 ) Entry Plasmids Protein fragment CM PDH 1 PSQC1 T/1–373 (T-protein) 130 98 2 PSQC2 T/93–373 0 25.2 3 PSQC3 T/96–373 0 55.0 4 PSQC4 T/101–373 0 0 5 PSQC5 T/93–277 0 0 6 PSQC6 T/93–297 0 0 7 PSQC7 T/93–316 0 0 8 PSQC8 T/93–336 0 0 9 pSQC24 T/1–88 1.8 0 10 pSQC9 T/1–94 11.4 0 11 pSQC10 T/1–108 8.1 0 12 pSQC11 T/1–276 10.1 0 13 pSQC12 T/1–296 7.9 0 14 pSQC13 T/1–316 9.2 0 15 pSQC14 T/1–336 8.8 0 760 S. Chen et al.(Eur. J. Biochem. 270) Ó FEBS 2003 T/1–336 was mildly elevated in the presence of Tyr at concentrations up to 10 l M . In contrast, the PDH activity in fragments T/93–373 and 96/373 was inhibited in the presence of Tyr, with 50% inhibition of activity in each protein fragment observed at 25 ± 5 l M Tyr. Molecular mass estimation and subunit association analysis The calculated molecular mass values for T/1–94 (12.5 kDa) and T/1–108 (14 kDa) agreed well with values obtained from SDS/PAGE using standard molecular mass markers (data not shown). Gel exclusion HPLC analysis was used to identify the molecular mass of the two fragments under native conditions. Using a standard curve based on the retention times and log molecular masses of four known proteins (Table 4), molecular masses for T/1–94 and T/93– 373 were calculated to be 25 kDa and 63 kDa, respectively, indicating that both fragments were dimers. As the molecular mass of the T-protein exceeded the effective range of gel exclusion HPLC analysis, chemical cross-linking was used to identify the state of the T-protein under native conditions (Fig. 4). SDS/PAGE analysis after cross-linking indicated that the native T-protein was a dimer, having a molecular mass of 85 kDa. Discussion The E. coli T- and P-proteins share numerous structural and kinetic similarities. Besides being native dimers (com- posed of subunits of similar M r values), both bifunctional catalysts are subject to end-product inhibition (by Tyr and Phe, respectively) induced by the aggregation of dimers into higher oligomers. Feedback inhibition in each case more strongly affects the second, prephenate-processing, enzyme (PDH and PDT, respectively). Several lines of evidence indicate that the major difference between the T- and P-proteins is the spatial and functional relationship between the two catalytic activities in each bifunctional enzyme. Earlier studies from these laboratories established that the CM, PDT, and regulatory functions of the E. coli P-protein reside in discrete, separable domains that can be subcloned and expressed [5]. In the case of the E. coli T-protein, several previous kinetic studies suggested interdependent, and perhaps overlapping [8] or closely proximal [9], CM and PDH active sites. The interdependence of the catalytic sites in the T-protein was first noted by Koch et al. who compared the rates of the CM and PDH reactions and observed a distinct lag phase in the latter process [16]. Furthermore, levels of free prephenate accumulating in the reaction mixture could not account for the observed rate of the PDH reaction, further suggesting interactions between the CM and PDH sites. Koch et al. also observed that the inhibition constant (K i ) for prephenate closely paralleled its K m value for the PDH reaction, and concluded that the CM and PDH-catalyzed reactions shared a common prephenate binding site on the T-protein. Subsequently, Heyde and Morrison noted that NAD + , the cofactor required for PDH activity, also boosted CM activity, while chorismate enhanced PDH activity [8]. The present study represents the first systematic effort to identify amino acid sequences within the T-protein that, when expressed as discrete fragments, displayed either CM or PDH activity. The main goal of the study was to learn whether CM or PDH activity might be separated into individual domains of the T-protein. A further goal of the study was to ascertain whether feedback inhibition by Tyr might also involve a discrete region of the T-protein. The established domain relationships in the P-protein suggested that a T-protein fragment embodying the N-terminus and the first 90–100 residues might exhibit CM activity. A modest level of sequence similarity (22 of the first 56 residues are identical [2]) in the N-terminal regions of the T- and P-proteins further supported this conclusion, although potential differences in secondary structure between the two proteins complicated any analysis based strictly on sequence comparison. The results of limited digestion of the T-protein using papain consistently affor- ded a pattern of fragments having principal bands at molecular masses 10 and 30 kDa. N-terminal sequence analysis indicated that the two fragments corresponded to residues 1–92 and 93–373, respectively. The finding that the smaller, 10 kDa fragment was somewhat resistant to proteolysis (Fig. 3) also lent credence to the possibility that it existed as a separately folded domain in the T-protein. The T-protein has been reported to be quite unstable in crude cell extracts [17], although stabilization of pure T-protein by prephenate or Tyr has been noted [16]. Heyde and Morrison observed that the T-protein exhibited poor stability when stored in dilute solution, causing the ratio of Fig. 4. The T-protein was cross-linked by 2% glutaraldehyde at 2.5 lgÆmL )1 of T-protein for 22 h. Lane 1, ladder; lane 2, T-protein control; lane 3, T-protein after cross-linking. Table 4. HPLC retention times and molecular masses of T-protein fragments and standards. Protein Retention time (min) Molecular mass (kDa) Aggregation state BSA 10.75 67 – Chicken ovalbumin 11.8 44 – Equine myoglobin 15.3 17 – CM1-94 13.2 25 Dimer PDH93-373 11.0 63 Dimer Ó FEBS 2003 E. coli T-protein catalytic and regulatory domains (Eur. J. Biochem. 270) 761 CM to PDH activity to vary from 0.8 to 1.2 between preparations [8]. It should be noted that the E. coli T-protein has been reported to be quite sensitive to both storage and aging [18]. The present study used T-protein expressed with a C-terminal His-tag to simplify purification. Chemical cross-linking experiments confirmed its dimeric structure under native conditions (Fig. 4) and its catalytic profile matched the wild-type protein. However, the stability of the His-tag labelled T-protein remained a problem. PDH activity deteriorated particularly rapidly during storage (Fig. 1), whereas significant levels of CM activity were retained. Taken together with results from limited proteo- lysis experiments, the data suggested that the region of the T-protein associated with PDH catalysis was more loosely packed, and hence more easily denatured, than the corres- ponding domain or residues associated with CM activity. His-tagged forms of the E. coli T-protein and 14 fragments thereof were successfully expressed and purified by affinity chromatography. Screening of those fragments for enzymatic activity (Table 3) indicated that neither the CM nor the PDH active site could be expressed in fully functional form as a discrete, contiguous subregion of the T-protein. Based on the seven fragments that displayed CM activity, residues 1–88 appeared to be essential for CM catalysis. While CM activity was enhanced by including the additional residues, 89–94, the most active fragment displayed only 8% of WT T-protein activity. Surprisingly, a stepwise increase in the fragment length (T/1–108, T/1– 276, T/1–296, T/1–316, T/1–336) did not increase CM activity. Several possible explanations were considered for the consistently low levels of mutase activity. The association of engineered fragments into homodimers, shown to be important in the monofunctional mutase derived from the E. coli P-protein, was confirmed in the case of T/1–94 by gel exclusion HPLC (Table 3). Contamination of the purified fragments by low levels of WT T-protein was ruled out by the absence of any corresponding PDH activity (Table 3). If the organization of the CM and PDH/PDT active sites in the T and P-proteins were similar, then a heterodimeric enzyme displaying CM but not PDH activity might plausibly arise by the complexation of one His-tagged fragment with one WT T-protein chain. This possibility seemed remote for two reasons. Because the cloned fragment was expressed at much higher concentrations compared to the native T-protein, any suspect heterodimer would have represented a very small amount of the protein. Moreover, analysis of each mutase-active fragment by SDS/PAGE at high gel loading levels revealed no higher molecular mass band matching the T-protein or corres- ponding heterodimer. The low mutase activity of the N-terminal fragments (Table 3) indicated that a discrete, fully active CM subdomain comprising contiguous T-protein residues could not be expressed, showing that a catalytically efficient CM active site required most, if not all, of the T-protein. An earlier report by Christendat et al.[15] indicated that mutagenesis of several residues in the dehydrogenase portion of the T-protein significantly affected CM activity, either by reducing K cat (His189Asn) or elevating K m (His239Asn, His245Asn). The findings reported here suggest that additional amino acids in the PDH domain, extending beyond residue 336 effect mutase activity. Proper CM function may be disrupted by poor substrate binding, as has been noted with the His239 and His245 mutants. Likewise, the series of N-terminal fragments (entries 9–15, Table 3) may have structurally altered or incomplete PDH substrate binding sites that cause poor substrate binding. If, as has been suggested [16], prephenate undergoes transfer from the product-binding pocket of the CM site to the substrate-binding pocket of the PDH site, then the weak CM activity of the CM fragments might be due to slow product release or trapping of prephenate on the truncated protein. In contrast, fragments of the T-protein could be prepared that contained catalytically competent, monofunctional dehydrogenases with the requisite NAD + binding sites. Two C-terminal sequences lacking approximately one- quarter of the T-protein’s N-terminal region were expressed (T/93–373 and T/96–373; entries 2–3, Table 3) that dis- played significant levels of PDH activity, but no CM activity. Xia et al. showed that a similar, monofunctional PDH domain could be prepared from the corresponding bifunctional protein in Erwinia herbicola by deleting residues 1–37 [19]. Earlier studies on the E. coli T-protein had implicated His197 as a key catalytic residue in PDH activity [15] and Arg294 in prephenate binding [20]. Both of these residues were included in the sequences of the two PDH- active fragments. Fragment T/101–373 (entry 4, Table 3) was devoid of PDH activity, suggesting that one or more residues in the 97–100 region may play an important role in catalysis. Of the fragments displaying monofunctional PDH acti- vity, analysis of one (T/93–373) by gel exclusion HPLC showed it to be a homodimer (Table 4). As the CM-active fragment T/1–94 was also a homodimer, these data indi- cated that noncovalent interactions resulting in T-protein dimerization appeared to be present in both the CM and PDH domains, unlike the P-protein, in which dimerizing interactions occurred only in the N-terminal region. Sam- ples of both T/93–373 and T/96–373 retained > 95% of their activity when stored for 7 days at )70 °Cand reassayed. However, both fragments underwent denatura- tion after prolonged storage (3–4 months at )20 °C), with complete loss of activity. With an N-terminal CM site joined to a PDH domain, the overall layout of chorismate and prephenate processing sites in the T-protein resembled that of the P-protein. However, results from the present study showed that the organization of the structural domains responsible for end product inhibition differed substantially in the two bifunctional proteins. Whereas the C-terminal 100 residues of the P-protein constituted a discrete Phe-binding domain, T-protein fragment analysis indicated that tyrosine binding and feedback inhibition could not be attributed to a structural domain that was separate from the CM and PDH domains. Initial attempts to pinpoint the C-terminal boundary of the PDH domain established that even minor deletions of C-terminal residues resulted in complete loss of PDH activity (entries 5–8, Table 3). Corresponding residue deletions in the P-protein did not diminish PDT activity. 762 S. Chen et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Because of the absence of a discrete regulatory domain in the T–protein, the interaction of various fragments with Tyr was investigated to determine whether the Tyr binding site overlapped with one or more catalytic domains in the T-protein. Tyr had no effect on the low CM activity observed in fragments T/1–88, T/1–94, T/1–108, T/1–276, T/1–296, and T/1–316. However, the CM activity of fragment T/1–336 was mildly enhanced at low Tyr concentrations (up to 10 l M ). A similar activation of CM activity in the WT T-protein was first observed by Christopherson at up to 300 l M Tyr [21] for which no mechanistic rationale has been proposed. The fact that activation by Tyr was weaker in T/1–336 suggested that the C-terminal 30 residues of the T-protein affected Tyr binding, and perhaps contributed to an allosteric effect on CM. Overall, the behavior of N-terminal fragments listed in Table 3 towards Tyr consistently indicated that the locus of Tyr binding included residues near the C-terminus of the T-protein. In agreement with that prediction, Tyr had a pronounced inhibitory effect on PDH-active fragments T/93–373 and T/96–373. In each case, 50% inhibition of enzyme activity was observed at 25 ± 5 l M , which agreed with the IC 50 value of 20 l M first reported by Koch et al.fortheWT T-protein [22]. Overall, these findings indicated that Tyr binding coincided with the region of the T-protein princi- pally associated with PDH activity, and provide a physical basis for the observation of Christopherson [21] that Tyr exerted a more pronounced effect on PDH activity than on CM activity. Koch et al. [16] had earlier proposed a form of sequential feedback inhibition in which Tyr acted primarily to inhibit PDH, resulting in an accumulation of prephenate that, in turn, inhibited CM. That picture is consistent with the physical layout of catalytic and binding sites that emerges from the T-protein fragment studies presented here. The domain mapping studies reported here, based on 14 T-protein fragments, indicated that CM and PDH were separable into independent enzymatic sites, although the efficiency of the CM-active fragments was considerably diminished when compared to the native T-protein. Acknowledgements This work was supported by grants from the National Institutes of Health (GM 24054, to BG) and the Department of Energy (DE-F G02-84ER13233, to DBW). References 1. Koch, G.L., Shaw, D.C. & Gibson, F. (1971) Characterisation of the subunits of chorismate mutase-prephenate dehydrogenase from E. coli K12. Biochim. Biophys. Acta 229, 805–812. 2. Haslam, E. (1993) Shikimic Acid Metabolism and Metabolites. John Wiley & Sons, New York, USA. 3. Hudson, G.S., Howlett, G.J. & Davidson, B.E. (1983) The binding of tyrosine and NAD + to chorismate mutase/prephenate dehy- drogenase from Escherichia coli K12 and the effects of these lig- ands on the activity and self association of the enzyme. J. Biol. Chem. 258, 3114–3120. 4. Pohnert, G., Zhang, S., Husain, A., Wilson, D.B. & Ganem, B. (1999) Regulation of phenylalanine biosynthesis. Calorimetric studies on the E. coli P-protein and its regulatory domain. Bio- chemistry 38, 12212–12217. 5. Zhang,S.,Pohnert,G.,Kongsaeree,P.,Wilson,D.B.,Clardy,J.& Ganem, B. (1998) Chorismate mutase-prephenate dehydratase from Escherichia coli: study of catalytic and regulatory domains using genetically engineered proteins. J. Biol. Chem. 273, 6248– 6253. 6. Zhang, S., Wilson, D.B. & Ganem, B. (2000) Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagen- esis of the Escherichia coli P-protein dehydratase domain. Biochemistry 39, 4722–4728. 7. Lee, A.Y., Stewart, J.D., Clardy, J. & Ganem, B. (1995) New insight into the catalytic mechanism of chorismate mutases from structural studies. Chem. Biol. 2, 195–203. 8. Heyde, E. & Morrison, J.F. (1978) Kinetic studies on the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes. Biochemistry 17, 1573–1580. 9. Turnbull, J. & Morrison, J.F. (1990) Chorismate mutase- prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites. Biochemistry 29, 10255–10261. 10. Turnbull, J., Cleland, W.W. & Morrison, J.F. (1991) pH dependency of the reactions catalyzed by chorismate mutase- prephenate dehydrogenase from Escherichia coli. Biochemistry 30, 7777–7782. 11. Christopherson, R.I. (1997) Partial inactivation of chorismate mutase-prephenate dehydrogenase from Escherichia coli in the presence of analogs of chorismate. Int. J. Biochem. Cell Biol. 29, 589–594. 12. Vincent, S., Chen, S., Wilson, D.B. & Ganem, B. (2002) Probing the overlap of chorismate mutase and prephenate dehydrogenase sites in the Escherichia coli T-protein: a dehydrogenase-selective inhibitor. Bioorg.Med.Chem.Lett.12, 929–931. 13. Zurawski, G., Brown, K., Killingly, D. & Yanofsky, C. (1978) Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli. Proc. Natl Acad. Sci. USA 75, 4271– 4275. 14. Davidson, B.E. & Hudson, G.S. (1987) Chorismate mutase- prephenate dehydrogenase from Escherichia coli. Methods Enzymol. 142, 440–450. 15. Christendat, D., Saridakis, V.C. & Turnbull, J.L. (1998) Use of site-directed mutagenesis to identify residues specific for each reaction catalyzed by chorismate mutase-prephenate dehydro- genase from Escherichia coli. Biochemistry 37, 15703–15712. 16. Koch, G.L.E., Shaw, D.C. & Gibson, F. (1972) Studies on the relationship between the active sites of chorismate mutase- prephenate dehydrogenase from Escherichia coli or Aerobacter aerogenes. Biochim. Biophys. Acta 258, 719–730. 17. Llewellyn, D.J. & Smith, G.D. (1979) Study of chorismate mutase- prephenate dehydrogenase in crude cell extracts of Escherichia coli. Biochemistry 18, 4707–4714. 18. Dopheide, T.A.A., Crewther, P. & Davidson, B.E. (1972) Choris- mate mutase-prephenate dehydratase from Escherichia coli K12. J. Biol. Chem. 247, 4447–4452. 19. Xia, T., Zhao, G., Fischer, R.S. & Jensen, R.A. (1992) J. General Microbiol. 138, 1309–1316. 20. Christendat, D. & Turnbull, J.L. (1999) Identifying groups involved in the binding of prephenate to prephenate dehydro- genase from Escherichia coli. Biochemistry 38, 4782–4793. 21. Christopherson, R.I. (1985) Chorismate mutase-prephenate dehydrogenase from Escherichia coli: cooperative effects and inhibition by 1-tyrosine. Arch. Biochem. Biophys. 240, 646–654. 22. Koch, G.L.E., Shaw, D.C. & Gibson, F. (1971) The purification and characterisation of chorismate mutase-prephenate dehydro- genase from Escherichia coli. Biochim. Biophys. Acta 229, 795– 812. Ó FEBS 2003 E. coli T-protein catalytic and regulatory domains (Eur. J. Biochem. 270) 763 . Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein Shuqing Chen 1, *, Sarah Vincent 2 , David B. Wilson 1 and Bruce Ganem 2 1 Department of. (1983) The binding of tyrosine and NAD + to chorismate mutase/ prephenate dehy- drogenase from Escherichia coli K12 and the effects of these lig- ands on the activity and self association of the enzyme on the E. coli T-protein had implicated His197 as a key catalytic residue in PDH activity [15] and Arg294 in prephenate binding [20]. Both of these residues were included in the sequences of the