From the Section of Biochemistry, Molecular and
Cellular Biology and the § Department of Chemistry, Baker
Laboratory, Cornell University, Ithaca, New York 14853-1301
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ABSTRACT |
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The bifunctional P-protein, which plays a central role in Escherichia coli phenylalanine biosynthesis, contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by phenylalanine). Six genes coding for P-protein domains or subdomains were constructed and successfully expressed. Proteins containing residues 1-285 and residues 1-300 retained full mutase and dehydratase activity, but exhibited no feedback inhibition. Proteins containing residues 101-386 and residues 101-300 retained full dehydratase activity, but lacked mutase activity. Fluorescence emission spectra and binding assays indicated that residues 286-386 were crucial for phenylalanine binding. The mutase (residues 1-109), dehydratase (residues 101-285), and regulatory (residues 286-386) activities were thus shown to reside in discrete domains of the P-protein. Both the mutase domain and the native P-protein formed dimers. Deletion of the mutase domain diminished phenylalanine binding to the regulatory site as well as prephenate binding to the dehydratase domain, both through cooperative effects. Besides eliminating feedback inhibition, removal of the R-domain decreased the affinity of chorismate mutase for chorismate.
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INTRODUCTION |
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Proteins employ a variety of regulatory mechanisms to achieve functional control, virtually all of which involve cooperative interactions between ligand binding sites (1). Understanding the detailed structural principles involved in allosteric interactions and other cooperative phenomena has become increasingly important in the rational design of new proteins with specifically engineered properties. A number of important applications can be envisioned for such proteins, including their use as novel molecular detection systems for industrial and biotechnological processes and as sophisticated biosensors in therapeutic monitoring and in biomedical devices (2).
Several feedback control mechanisms are known in the biosynthesis of the aromatic amino acids, phenylalanine (Phe)1 and tyrosine (Tyr), via the shikimic acid pathway. Both Phe and Tyr are produced from chorismic acid, a key intermediate in the shikimate pathway whose metabolism branches in five different directions (3). Each of the five branch point enzymes must be carefully regulated to partition chorismate properly to various downstream intermediates.
In the first committed step to Phe and Tyr, chorismate undergoes a
Claisen rearrangement to prephenate catalyzed by chorismate mutase (CM,
EC 5.4.99.5). Prephenate can undergo either decarboxylation/dehydration to phenylpyruvate catalyzed by prephenate dehydratase (PDT, EC 4.2.1.51) or decarboxylation/dehydrogenation to
p-hydroxyphenylpyruvate catalyzed by prephenate
dehydrogenase. Transamination of each -ketoacid produces the
respective
-amino acid. In an alternative pathway, prephenate can
first form arogenic acid and subsequently be converted to Phe and Tyr
(4).
Procaryotes, eucaryotes, and higher plants may use either route to Phe and Tyr, and many organisms have achieved the desired levels of regulation by evolving multiple isozyme systems. For example, CMs have been identified that are monofunctional with allosteric control, monofunctional lacking allosteric control, or bifunctional (5). To date, three bifunctional CMs are known in which mutase activity is coupled with PDT, prephenate dehydrogenase, or 3-deoxyarabinoheptulosonate-7-phosphate synthase (5).
To understand the various domain interactions and allosteric effects in such bifunctional proteins, we elected to study the P-protein of Escherichia coli, in which CM and PDT are coupled (1, 6). Both activities are subject to feedback inhibition by Phe (7). The P-protein is encoded by the PheA gene and contains 386 amino acids with a molecular mass of 43 kDa (8, 9). The catalytically active form of the enzyme is a homodimer (10, 11); however, increasing Phe concentrations shift the P-protein from a dimer to a mixture of dimer, tetramer, and higher order species, suggesting cooperative homotropic interactions between Phe binding sites (10-12).
Our approach, which involved expressing discrete CM, PDT, and R-domains from genetically engineered fragments of PheA, was guided by several considerations. First, we hoped to isolate and identify the individual binding sites for the P-protein's substrates and products to better understand the interactions between CM, PDT, and their effectors. Second, while earlier chemical modifications (13, 14), kinetic studies (15), and genetic mutations (16-18) suggested that the CM, PDT, and R-domains were relatively distinct, it was unclear to what extent the full, continuous polypeptide backbone would be necessary for correct folding, expression, and activity. Indeed, some degree of domain interconnection has been noted (16, 19), which could be probed in detail using the recombinant approach. Third, we expected that specifically engineered domains might shed light on the detailed molecular interactions involved in dimerization and in the formation of Phe-induced higher order aggregates.
Earlier we successfully cloned, expressed, and crystallized the NH2-terminal 109 residues of the E. coli P-protein, a fully active CM domain with no PDT or Phe binding activity (20, 21). Like the native P-protein, it formed a stable dimer. It was therefore of interest to create a smaller, monofunctional PDT domain to help understand the molecular interactions involved in feedback inhibition and higher order aggregation. Also of interest, from the perspective of designing biosensors for amino acids (2), was the possibility of excising a fully functional R-domain from the P-protein backbone. Here we report the use of genetic engineering to map the P-protein's PDT and R-domains and describe how domain interactions affect higher order structure.
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EXPERIMENTAL PROCEDURES |
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Materials-- Unless indicated otherwise, all chemicals were purchased from Sigma, and biochemicals were obtained from New England Biolabs. L-(4-3H)-Phenylalanine (26.0 Ci/mmol) was obtained from Amersham Corp. and diluted with cold Phe. Polyethylene equilibrium dialysis cells having a volume of 0.2 ml were used for equilibrium dialysis (22).
Strains-- E. coli strain KS474 (relevant genotype: degP41{DPstI-KanI}) was used as the host for cloning, plasmid isolation and expression (23). Unless indicated, strains harboring plasmids were grown in either M9 medium, Luria broth, or Luria agar plates all containing 100 µg/ml of ampicillin. E. coli strain GI724, obtained from Invitrogen, Inc., was used as the host for making Thio-R.
Proteolysis of the P-protein-- The P-protein was purified (24) and partially digested by adding 1 µl of 0.2 mg/ml elastase in reaction buffer (50 mM Tris, pH 8.3, 5 mM EDTA) to 25 µg of purified P-protein in 49 µl of reaction buffer with or without 1 mM Phe. Gel electrophoresis indicated that proteins of 34 and 32 kDa, respectively, were formed when digestion occurred in the presence or absence of Phe.
Recombinant DNA Manipulations and Plasmids-- Six plasmids (pSZ41, pSZ43, pSZ44, pSZ50, pSZ70, and pSZ85) were constructed from pJS1, which carries the pheA gene in pUC18 as described previously (24). To delete the potential COOH-terminal domain, two oligonucleotides (33-mer and 31-mer) were synthesized that introduced a stop codon and a HindIII site after residue 300 or 285, respectively, and used as reverse primers for PCR. The M13/pUC universal primer 1233 (from Biolabs) was the forward primer using pJS1 as a template. PCR was carried out at 95 °C (1 min), 55 °C (1 min), and 72 °C (1.5 min) with 35 cycles. The resulting PCR products and pUC18 were cut with EcoRI and HindIII, and the desired DNA fragments were isolated on a 1.0% agarose gel, ligated, and transformed into KS474 by the TSS method (25). The resulting transformants were screened by restriction site analysis. The positive plasmids were named pSZ44 and pSZ70, respectively. For the clones removing the NH2-terminal domain of the P-protein, a 29-mer synthetic oligonucleotide with a SalI site created between the ribosome binding site and the initial codon of pheA was used as a reverse primer for PCR as described above. After treatment with EcoRI and SalI, the PCR product containing 620 base pairs of the pheA promoter region with a SalI site at the 3' end was isolated.
Two oligonucleotides that initiate translation at residues 101 and 114 of the P-protein were synthesized containing an initiation codon preceded by a SalI site. These were used as forward primers in PCR with the M13/pUC universal primer 1224 (Biolabs) as a reverse primer and pJS1 as the template. The PCR products were treated with SalI and HindIII and ligated with the above 620-base pair fragment into a pUC18 fragment with EcoRI and HindIII ends. The resulting positive plasmids were pSZ43 and pSZ41, respectively. A PCR fragment encoding residues 100~300 of the P-protein was obtained using pSZ43 as a template with the above 33-mer oligonucleotide as a reverse primer and universal primer 1233 as a forward primer and cloned into pUC18 at the EcoRI and HindIII sites to yield pSZ50. A 490-base pair fragment encoding Arg279 to Thr386 was created by PCR using 5' CGATTTGTGGTGTTGGCGCGT 3' as a forward primer and Universal primer 1224 as a reverse primer with pJS1 as template. The resulting PCR product was cut with PstI, ligated to the SmaI and PstI sites of the pTrxFur plasmid (Invitrogen), and transformed into GI724 to give pSZ85, which coded for Thio-R. All six plasmids containing partial pheA genes were sequenced on an Applied Biosystems model 373A DNA sequencer in the Cornell Biotechnology Facility. The nucleotide sequences and deduced peptide sequences were analyzed with the DNASTAR Program.Expression and Crude Extract Preparation-- Cell pellets and crude cell extracts were screened by Western blotting to confirm expression in E. coli. Proteins were separated by 15% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and detected with anti-P-protein antibody as described (26). Cell extracts from each sample were prepared as before (24) and were tested for CM, PDT, and Phe binding activities using a pUC18 cell extract as a negative control. When overexpressed from pSZ85, Thio-R underwent partial proteolysis to liberate 12- and 13-kDa fragments during workup of the crude extracts. The 12-kDa fragment, designated R12, was shown by NH2-terminal sequencing to contain residues 286-386 of the P-protein. The 13-kDa fragment, produced in equal amounts, was presumed to be thioredoxin.
Purification of the Engineered Proteins-- All proteins were purified by modifying a published procedure (24). After the Q-Sepharose column, each protein was loaded on a Poros HQ column (0.5 × 5 cm) preequilibrated with 25 mM Tris, pH 8.8, + 10% glycerol and eluted with a linear gradient (30-fold of column volume) of 0~100 mM NaCl in equilibration buffer. Active fractions were pooled and concentrated by centrifugation using a Centricon-10 (from Amicon). Thio-R and R12 were purified on a Thio-Bond Resin affinity column (Invitrogen), and R12 was further purified by gel filtration on an ACA44 column. Protein purity and subunit molecular weight were determined on a 15% SDS-polyacrylamide gel electrophoresis and an isoelectric focusing gel (Islab). Protein concentrations were determined by the Bradford method (27) using bovine serum albumin as a standard.
Molecular Mass Estimation and NH2-terminal Analysis-- The molecular masses of all purified proteins were estimated by gel filtration on a Waters Protein Pak Glass 300SW HPLC column. Molecular weights of each enzyme were estimated from elution volumes (relative to standards of known molecular weight run under the same conditions). Purified proteins were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically blotted onto an Immobilon-P membrane (polyvinylidene difluoride membrane from Millipore) as described (28). NH2-terminal sequences were determined using an Applied Biosystems 470A protein sequenator.
Fluorescence Measurements-- Fluorescence measurements were performed on an Aminco SCM 8000C spectrofluorimeter equipped with a xenon light source, using an excitation wavelength of 295 nm. The emission was scanned from 305 to 400 nm with the slit widths for excitation and emission set at 4 nm. Protein concentrations were kept at about 2.5 µM in 10 mM Tris, pH 8.0, and dilution effects from the Phe titrations were negligible. Measurements were performed at 37 °C, and all spectra were corrected for background and Raman scattering by subtracting the buffer signal.
Phe Binding Assay-- Filtration binding assays were performed by measuring enzyme bound Phe adsorbed to a nitrocellulose filter following a published procedure (29). The binding and wash buffer were 20 mM Tris, pH 8.2, 10 mM EDTA, 0.01% bovine serum albumin, 20 mM mercaptoethanol, and 100 mM KCl. Titration of the proteins with [3H]Phe was run at a constant enzyme concentration of 4 µM incubated with a ratio of [3H]Phe/protein from 0.1 to 10, while titration of [3H]Phe with protein was run at 0.096 µM [3H]Phe (2.4 pmol of Phe/filter = 28,000 cpm) with protein concentrations from 0.2 µM to 16 µM. Equilibrium dialysis experiments were conducted by equilibrating protein (1.2 nmol) against [3H]Phe (36 µM, final radioactivity of 2.5 µCi/ml) at 30 °C for 16 h, then determining the Phe concentration on each side of the dialysis cell by scintillation counting. The free and bound Phe as well as the Kd value were calculated. The protein concentration in both compartments was tested by an activity assay to ensure no leakage occurred during equilibrium.
Enzyme Assay and Kinetic Studies-- Chorismate mutase activity was assayed by monitoring the conversion of chorismate to prephenate; PDT activity was measured by the conversion of prephenate to phenylpyruvate as described previously (30). One unit of enzyme was defined as the amount of enzyme required to form 1 µmol of product/min at 37 °C. Substrate and effector concentrations are described in the individual experiments. Kinetic parameters were determined with the curve fitting options in the KaleidaGraph program (Abelbeck Software).
Analysis of PDT32 in the presence of Phe indicated mixed-type inhibition kinetics (a form of noncompetitive inhibition) described by the equation v/Vmax = S/[Ks(1 + I/Ki)+ S(1 + I/Thermostability Assay-- Purified proteins (10 µmol/liter in 20 mM Tris, pH 8.0, 10% glycerol, 50 mM NaCl, and 5 mM mercaptoethanol with or without 1 mM Phe) were incubated at 25-65 °C for 1 h. The temperature at which 50% (CM or PDT) activity remained after preincubation for 1 h was measured to ascertain the stability of each enzyme.
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RESULTS |
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Expression, Purification, and Characterization of the Engineered Enzymes-- Six new plasmids (Table I) were constructed, sequenced, and expressed in E. coli KS474 and GI724 (Invitrogen), and the corresponding expressed proteins were designated with abbreviations indicating both function and size. Thus, PDT22 and PDT32 refer to 22- and 32-kDa proteins exhibiting only PDT activity, whereas P*300 and P*285 designate COOH-terminal truncated P-proteins exhibiting both CM and PDT activity, but no feedback inhibition by Phe.
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Phe-Protein Interactions-- Fluorescence properties of the intact P-protein changed significantly in the presence of Phe. The intrinsic fluorescence emission intensity increased and the emission maximum was shifted from 341 to 338 nm in the presence of 1 mM Phe (Fig. 2A). The increase in emission intensity at 340 nm was monitored in titration experiments with Phe and correlated well with the inhibition of PDT activity. In the presence of 1 mM Phe, the emission spectra of PDT32 and R12 displayed a shift to higher wavelengths (335 and 341 nm to 338 and 344 nm, respectively; Fig. 2B). The relative fluorescence intensity increased as well. Phenylalanine had no effect on the fluorimetric properties of P*300, P*285, and PDT22, which contained Trp226 as the only fluorescent amino acid (emission maxima 332-334 nm) (Fig. 2A). The fluorescence emission maximum of Thio-R (338 nm) was shifted up ~0.5 nm after addition of 1 mM Phe. Control experiments with D-Phe and with increased concentrations of L-leucine and L-tyrosine had no effect on the fluorimetric properties of the P-protein and the different domains.
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Activity and Kinetic Characterization-- The specific activities of both CM and PDT were determined for PDT22, PDT32, P*285, and P*300 at 1 mM chorismate and 0.5 mM prephenate (Table III) and were slightly lower than previously reported (CM = 45 units/mg; PDT = 22 units/mg) (24). All four enzymes retained about 90% of the PDT activity observed in the P-protein. As expected, the PDT32 and PDT22 proteins lacked CM activity.
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Thermal Inactivation Assay-- All four engineered enzymes, whether mono- or bifunctional, displayed somewhat better thermal stability than the P-protein. Fifty percent inactivation in 1 h was observed at 54 °C for the P-protein, 58 °C for P*285 and P*300 and 62 °C for PDT32 and PDT22. Studies with P, P*285, and P*300 revealed little difference in the thermal inactivation of CM and PDT domains (Figs. 5, A and B). In the case of PDT32, the presence of Phe retarded thermal inactivation, although not as effectively as with the P-protein (Fig. 5C). The presence of Phe also improved the thermostability of the P-protein's CM domain (Fig. 5B).
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DISCUSSION |
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Our results indicate that the PDT domain of the P-protein, like the CM domain (20, 21), could be expressed as a fully active, monofunctional enzyme. Kinetic data on PDT22 (residues 101-300) and P*285 (residues 1-285), together with the absence of PDT activity in R30 (residues 114-386), located the P-protein's dehydratase domain in residues 101-285.
When separated, the individual CM and PDT domains displayed greater robustness, as noted in thermostability assays (Fig. 5). Since smaller domains may refold more easily than larger, multidomain proteins, this apparent increase in stability might be attributed to more rapid renaturation of the smaller fragments upon cooling.
Both the CM and PDT domains, in their smallest active forms, were unaffected by Phe. This finding was consistent with earlier work of Backman and Ramaswamy (35), who noted that deletion of residues 338-386 in the E. coli P-protein also reduced feedback sensitivity to Phe without destroying catalytic activity. Xia et al. (18) reported that a truncated pheA construct from Erwinia herbicola lacking residues 301-387 lost allosteric control, but retained bifunctional catalytic competence.
Both the CM and R-domains exerted discrete effects on the overall structure of the P-protein. In the absence of Phe, proteins lacking the CM domain (PDT32, PDT22, R12) formed monomers, whereas proteins containing the CM domain (P-protein, P*285, P*300) formed dimers. The P*300 protein remained dimeric in the presence of Phe, implicating residues 301-386 in higher oligomer formation. Monomeric PDT32, which lacked the CM domain but retained the R-domain, formed dimers in the presence of Phe, but not tetramers and higher oligomers. We therefore concluded that the CM domain was responsible for dimerization of the P-protein and that the R-domain was responsible for higher order Phe-induced P-protein aggregates.
Using fluorimetric analysis to elucidate allosteric effects, a specific interaction of the R-domain with Phe was noted, although Phe binding was diminished in the absence of the catalytic domains. Earlier spectrophotometric studies had indicated changes upon Phe binding in the environment of the P-protein's tryptophan residues, Trp226 and/or Trp338 (36). More specific and sensitive fluorimetric measurements (Fig. 2A), demonstrated that the conformational changes were localized in the vicinity of Trp338. The interaction was specific for L-Phe and led to higher order aggregates of the P-protein, which were not observed with PDT32 and R12.
Feedback inhibition by Phe was more pronounced on PDT than on CM, with
inhibition of dehydratase activity in the P-protein increasing
sigmoidally with Phe concentration (7). Likewise PDT32 displayed a
mixed type of noncompetitive inhibition, with binding of Phe to the
R-domain causing a 9-fold lowering in the affinity of the dehydratase
active site for prephenate (Ki = 1950 µM). The addition of a CM inhibitor to the P-protein did not change Phe binding (Table II), suggesting that the mutase site had
little effect on the allosteric site. The value of the intrinsic Phe
binding constant (Kd) was ~5-fold higher for PDT32
than for the P-protein, which was attributed to the fact that the
P-protein formed a mixture of aggregation states in the presence of
Phe, each with a potentially different binding affinity.
Several P-protein domain interactions were noted. Removal of the CM domain diminished cooperative binding and affected both the R-domain (through weakened Phe binding) and the PDT domain (through weakened prephenate binding). Removal of the R-domain not only eliminated feedback inhibition, but also decreased the affinity of CM for chorismate. The full effect of feedback inhibition by Phe required a binding domain consisting of the P-protein's COOH-terminal 101 residues.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM 24054 (to B. G.) and CA 24487 (to J. C.) and Department of Energy Grant DE-F G02-84ER13233 (to D. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Received postdoctoral fellowship from the Fonds der Chemischen Industrie (Frankfurt, Germany).
To whom correspondence should be addressed. Tel.:
607-255-7360; Fax: 607-255-6318; E-mail: bg18{at}cornell.edu.
1 The abbreviations used are: Phe, L-phenylalanine; Tyr, L-tyrosine; CM, chorismate mutase; PDT, prephenate dehydratase; R-domain, regulatory domain; Thio-R, thioredoxin/R-domain fusion protein; PCR, polymerase chain reaction.
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REFERENCES |
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