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.. Enzyme assa
Trang 1Mapping of chorismate mutase and prephenate dehydrogenase
Shuqing Chen1,*, Sarah Vincent2, David B Wilson1and Bruce Ganem2
1
Department of Molecular Biology and Genetics and2Department 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 CMdomain, 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 CMand PDH domains were comparatively low, suggesting some cooper-ative interactions in the ncooper-ative state Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CMdomain
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
CMand 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 CMand PDH, and induces aggregation of the
T-protein [3] An analogous bifunctional protein in E coli,
known as the P-protein, contains CMand 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 CMin 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 CMand PDH reactions occurred at overlapping [8] or perhaps closely proximal [9] active sites Strong evidence for two separate CMand 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 CMactivity 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 CMactivity [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)
Trang 2of 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 coliBL21 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 NdeI and XhoI
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) at 37C 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 mMisopropyl b-D-thiogalactoside at
D660¼ 0.8 and growth at 30 C for 2.5 h, cells were collected by centrifugation at 10 000 g at 4C for 25 min Cell pellets were resuspended in cold binding buffer (5 mM
imidazole, 0.5MNaCl, 20 mMTris/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 lL of 0.1M NH4Ac, 0.004M EDTA, 0.01M cysteine (pH 6.8) and 0.4 lL of 0.1 mgÆmL)1 (1 : 50 ratio) or 2 lL of 0.01 mgÆmL)1 (1 : 1000 ratio) of papain were added The reaction was incubated at 37C and 10 lL 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 mM chorismate or 0.2 mM prephenate and 2 mM
NAD, respectively One unit of enzyme was defined as the amount of enzyme required to produce 1 lmol of product per minute at 37C 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
0 to 0.3 mM, with substrate concentrations ranging from 0
to 1 mMor 2 mMbased on the Kmvalue 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¢
Trang 3velocity (Vmax) and the Michaelis constant (Km) were
determined using standard rate equations in conjunction
with the curve fitting options in theKALEIDAGRAPHprogram
(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 mM Tris/
HCl, pH 8.0, 50 mM 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 mM KH2PO4/
K2HPO4 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 2MNaBH4/0.1MNaOH was
added to quench the reaction After standing at room
temperature for 20 min, 20 lL of 10% sodium
deoxycho-late in 0.1MNaOH 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 (30C),
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, and that for PDH was
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 (0.1M
sodium citrate : 10% glycerol : 1 mMdithiothreitol, pH 7.5)
or in assay buffer (0.1MMes, 0.051MN-ethylmorpholine, 0.01Mdiethanolamine, 1 mMEDTA, 1 mMdithiothreitol, 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-protein¼ 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 lM), but without NAD+
Digestions carried out in the presence of higher con-centrations of papain (papain : T-protein¼ 1 : 50) for limited periods of time revealed that the 30 kDa fragment
Fig 1 CM and PDH activity lost during storage.
Trang 4disappeared 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 CMand 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 CMactivity 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,
Km for CMactivity in T/1–88 and T/1–336 were
1.7 ± 0.1 mM and 2.4 ± 0.5 mM, respectively By
com-parison, Kmfor the T-protein was 0.23 mM None of the
fragments exhibiting CMactivity 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 CMactivity 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 CMactivity 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 CMactivity of fragments T/1–88, T/1–94 and T/1–108 was unaffected by Tyr at concentrations up to 300 lM The CMactivity 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
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 ofthe 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 ofT-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 ofcloned segments ofthe T-protein.
Enzyme activity (UÆmg)1) Entry Plasmids Protein fragment CMPDH
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
Trang 5T/1–336 was mildly elevated in the presence of Tyr at
concentrations up to 10 lM 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 lMTyr
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 Mrvalues), 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], CMand 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 CMand 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 CMand PDH sites Koch et al also observed that the inhibition constant (Ki) for prephenate closely paralleled its Km value for the PDH reaction, and concluded that the CMand 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 CMactivity, 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 CMor 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 CMactivity 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)1ofT-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 ofT-protein
fragments and standards.
Protein
Retention time (min)
Molecular mass (kDa)
Aggregation state
Chicken ovalbumin 11.8 44 –
Equine myoglobin 15.3 17 –
CM1-94 13.2 25 Dimer
PDH93-373 11.0 63 Dimer
Trang 6CMto 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 CMactivity 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 CMactivity
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
CMnor 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
CMactivity, residues 1–88 appeared to be essential for CM
catalysis While CMactivity 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 coliP-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 CMand PDH/PDT active sites in
the T and P-proteins were similar, then a heterodimeric
enzyme displaying CMbut 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 CMactive 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 CMactivity, either by reducing Kcat(His189Asn)
or elevating K (His239Asn, His245Asn) The findings
reported here suggest that additional amino acids in the PDH domain, extending beyond residue 336 effect mutase activity
Proper CMfunction 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 CMsite to the substrate-binding pocket of the PDH site, then the weak CMactivity of the CMfragments 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 CMand 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 C and 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 CMsite 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 CMand 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
Trang 7Because 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 CMactivity
observed in fragments T/1–88, T/1–94, T/1–108, T/1–276,
T/1–296, and T/1–316 However, the CMactivity of
fragment T/1–336 was mildly enhanced at low Tyr
concentrations (up to 10 lM) A similar activation of
CMactivity in the WT T-protein was first observed by
Christopherson at up to 300 lM 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 lM, which agreed with the IC50
value of 20 lM first reported by Koch et al for the WT
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
CMactivity 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 CMand 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 (GM24054, to BG) and the Department of Energy
(DE-F G02-84ER13233, to DBW).
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