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INTRODUCTION |
Tyrosine hydroxylase
(TH1) (tyrosine
3-monooxygenase, EC 1.14.16.2) is an eukaryotic enzyme catalyzing the
first and rate-limiting step in dopamine and other catecholamine
biosynthesis, i.e. the hydroxylation of the monophenol amino
acid L-tyrosine to produce the ortho-diphenol
L-dihydroxyphenylalanine (2, 3). The enzyme is active in
the presence of ferrous iron, O2, and a tetrahydrobiopterin co-substrate. A single gene encodes TH, which is required for embryonic
development and survival in mammals (4, 5). In vertebrates, TH activity
is exquisitely regulated at each step of its expression: control of
gene transcription, RNA alternative processing, mRNA stability, and
direct modulation of the enzyme by catecholamine feedback inhibition
and protein kinase activation (6-8).
In contrast, much less is known on the regulatory properties of
tyrosine hydroxylase in insects. Mutations in the Drosophila pale locus, which corresponds to DTH (9-11), result in
unpigmented embryos that are unable to hatch. It has been shown that
dopamine has a dual function in insects, acting as a neurotransmitter
in the central nervous system (12, 13) and as a precursor molecule required for pigmentation and hardening of the cuticle (14-16). The TH
enzyme is composed of a carboxyl-terminal catalytic domain and an
amino-terminal regulatory domain (17-21). The catalytic domain has
been well conserved in Drosophila TH (22). The regulatory domain is not conserved but contains a potential protein kinase A (PKA)
site occurring at Ser32 that is homologous to
Ser40, the major site of phosphorylation by PKA in
vertebrate TH (8). Two different forms of TH proteins have been found
in Drosophila melanogaster, which are produced through
alternative splicing of a single copy gene (1). The major form,
Drosophila TH Type II (DTH II), contains a very acidic
segment of 71 amino acids inserted in the regulatory domain close to
the PKA phosphorylation site. The two DTH isoforms are expressed in
distinct tissues; DTH I is specific of the nervous tissue, whereas DTH
II is widely expressed in non-nervous tissues (1). DTH II is strongly
expressed in the epidermis, or hypoderm, the single-layered epithelium
that covers the insect body and secretes the cuticle.
To compare the kinetic and regulatory properties of the two
Drosophila TH isoforms in vitro, we have
expressed each of these molecules as recombinant apoenzymes in a
bacterial expression system. Both isoforms were produced at a high
level and purified. We show here that the two enzymes differ in many of
their properties, including their pH profiles, iron requirements, and
Km values for
(6R)-5,6,7,8-tetrahydrobiopterin (BH4). In
addition, the two enzymes are differentially regulated by dopamine
feedback inhibition and activation by cAMP-dependent
phosphorylation. Our data suggest that the acidic extension in the
regulatory domain of DTH II endogenously activates the enzyme. The
structural difference between the two DTH isoforms could therefore have
a functional role and correspond to a differential regulation of
dopamine biosynthesis in nervous and non-nervous tissues of insects.
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EXPERIMENTAL PROCEDURES |
Construction of the DTH Expression Vectors--
The cDNA
clones pDTHcDNA1 and pDTHcDNA2, which encode DTH Type I and
Type II, respectively, were isolated previously (1). cDNA segments
containing the complete coding sequence of each DTH isoform were
subcloned into the Escherichia coli expression vector pET-11a (23). First, a NdeI site was introduced at
the translation start site of both cDNAs by site-directed
mutagenesis. The method we used was a modification of the three primer
PCR mutagenesis procedure. PCRs were carried out with Vent polymerase (New England Biolabs) in a 50-µl final volume as described by Marini
et al. (24) using 1 ng of the plasmid pDTHcDNA1 as a template. The primary reaction was performed with a 5' external sense
DTH oligonucleotide primer (OTH1', 5'-TTCGCCCTAAAGACTTGTGC) and an
internal mutagenesis antisense DTH primer with two mismatches (OTHm1,
5'-CGGCCATCATATGGTTTTGTGTG;
mismatches are bold, and the NdeI site is underlined). The
product of this PCR, a double-stranded 442-bp DNA segment intermediate,
was purified and used as a "megaprimer" in a second similar PCR
with a 3' external antisense DTH primer (OTH2,
5'-CAACAAAATCTCGTCCTCGGTGAGACC). The final 645-bp amplification product
was digested with XbaI and XhoI and inserted to
replace the corresponding nonmutated segment in pDTHcDNA1 and
pDTHcDNA2. Finally, NdeI-BclI cDNA
segments containing the coding sequence of the DTH isoforms Type
I and Type II were ligated to the vector pET-11a previously digested
with NdeI and BamHI to generate the recombinant
expression vectors pEDTHI and pEDTHII, respectively.
To replace Ser32 with Arg in both DTH isoforms, a
mutagenesis DTH sense primer was synthesized (OTHm2,
5'CGCCGTCGCCGCCTGGTGGAT). A double-stranded 131-bp DNA
segment intermediate was amplified from pDTHcDNA1 with the primers
OTH2 and OTHm2. This mutated 131-bp segment and the 442-bp segment
obtained previously with the primers OTH1' and OTHm1 were joined by a
second PCR. The final doubly mutated 645-bp amplification product was
used as described for the previous constructions to generate the
recombinant expression vectors pEDTHI(S32R) and pEDTHII(S32R). All
mutations were checked by PCR and confirmed by double-stranded DNA sequencing.
Expression of Recombinant DTH Isoforms--
DTH expression
vectors were introduced by electroporation into E. coli
BL21(DE3) cells (Novagen), which do not express the lon and
ompT proteases (23). Fresh cultures inoculated with a 1:100
dilution of a 10-11 h preculture were grown in M9ZB (23) plus 100 µg/ml carbenicillin for 3.5 h at 37 °C. Cells were
centrifuged and resuspended in the same volume of fresh medium. A
1-liter culture in M9ZB plus 100 µg/ml carbenicillin was inoculated
with 10 ml of these cells and grown for 2 h at 37 °C with
vigorous shaking until A600 was equal to
0.4-0.5. To minimize the formation of inclusion bodies, the culture
was then transferred at 18 °C with gentle shaking (125 rpm). DTH
expression was induced 30 min later by adding isopropyl
-D-thiogalactopyranoside to 1 mM. After overnight (13-14 h) incubation at 18 °C, cells were harvested by
centrifugation at 5000 × g for 15 min. Weighted
pellets (5.9-6.2 g) were washed once in ice-cold 0.3 M
sucrose, 0.1 mM EDTA, and 50 mM Tris-HCl, pH
7.5. Cells were then resuspended at 0.15-0.20 g/ml in the same
ice-cold buffer supplemented with 0.5 mM dithiothreitol, 5 µM leupeptin, 1 µM pepstatin, 0.5 mM phenylmethylsulfonylfluoride, 0.1 mg/ml lysozyme, and
0.1 mg/ml DNase. Cell lysis was completed by sonication or by using a
French press and checked by microscope examination. Insoluble proteins
were removed by centrifugation at 27,000 × g for 60 min at 4 °C, and the clear supernatant (bacterial soluble extract)
was complemented with glycerol to 10% (v/v) and either stored at
80 °C or used immediately for DTH purification.
Purification of Recombinant DTH Isoforms--
The procedure used
to purify the Drosophila tyrosine hydroxylase isoforms
expressed in E. coli was adapted from the method employed by
Daubner et al. (19) to purify the recombinant COOH-terminal domain of rat TH. All the enzyme purification steps were carried out at
4 °C. The soluble extract from 6 g of cells expressing either
DTH I or DTH II was applied to an XK16 column packed with 16 ml of
DEAE-Sepharose Fast Flow (Amersham Pharmacia Biotech) in column buffer
(50 mM Tris-HCl, pH 7.5, 10% glycerol (v/v), 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 µM leupeptin, 0.1 µM pepstatin). After
washing with 40 ml of column buffer at 0.5 ml/min, the bound proteins
were eluted by a 60-ml linear gradient of 0-0.675 M KCl in
the same buffer. TH activity was assayed, and the fractions corresponding to the peak of enzyme activity at about 0.4 M
KCl were pooled. Enzymes were then concentrated and further purified by
ammonium sulfate fractionation. Proteins precipitating between 32 and
40% saturation for DTH Type I and between 20 and 32% saturation for
DTH Type II were collected. The precipitate was solubilized in 40 ml of
column buffer and applied to a Mono-Q HR 5/5 column equilibrated in the
same buffer. After washing, the enzyme was eluted by a 30-ml linear
gradient of 0-0.85 M KCl at 0.5 ml/min. Fractions
containing the highest TH activity were pooled.
Protein and Western Blot Analysis--
Protein concentrations
were determined using the method of Bradford (25) with bovine serum
albumin as a standard. Protein electrophoresis was performed on 10%
polyacrylamide gels in the presence of 0.1% SDS-PAGE followed by
Coomassie Blue staining. Molecular weights were estimated by comparison
with protein standards (Perfect Protein Markers, Novagen). For Western
blot experiments, proteins from unstained gels were transferred to a
nitrocellulose sheet. The membrane was preincubated for 1 h in
10% nonfat dry milk in TBS (0.1% Tween 20, 500 mM NaCl,
20 mM Tris-HCl, pH 7.4) and then washed three times for 10 min in TBS. Drosophila tyrosine hydroxylase was probed with
a 1:5000 dilution of an affinity purified rabbit polyclonal anti-rat TH
antibody (Pel-Freez Biologicals) or a 1:1000 dilution of a rat
polyclonal antibody to Drosophila TH Type II-specific exons
C and D raised in the laboratory of Dr. Jay Hirsh (University of
Virginia). The antibodies were preadsorbed on bacterial proteins and
incubated with the blot for 1 h at 25 °C. The membrane was
washed as above and then incubated for 1 h with a 1:5000 dilution
of goat anti-rabbit or anti-rat IgG conjugated with horseradish
peroxidase (Jackson ImmunoResearch). Immunoreactive bands were
visualized by chemiluminescence with the ECL reagent (Amersham
Pharmacia Biotech). To remove bound antibodies, the membrane was
blocked in 5% nonfat dry milk in TBS for 10 min, incubated for 30 min
at 80 °C in stripping buffer (100 mM
-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8),
and washed three times for 10 min at room temperature in TBS.
Determination of TH Enzymatic Activity--
Tyrosine hydroxylase
activity was assayed by measuring the enzymatic release of tritium from
L-[3,5-3H]tyrosine. For kinetic studies, the
standard conditions for the assay were 50 mM K-Hepes, 50 µM L-tyrosine, 2.5 µCi/ml
L-[3,5-3H]tyrosine (50 Ci/mmol, Amersham), 20 µM BH4, 10 µM ferrous ammonium sulfate, 15 mM
-mercaptoethanol, 0.07 mg/ml catalase
(Sigma), with 0.1-0.5 µg of purified DTH at pH 7.0 in a volume of
0.1 ml. After a 2-3-min equilibration of the mixture at the assay
temperature (25 or 29 °C), the reaction was started by the addition
of BH4. Assays were stopped by the addition of 1 ml of
7.5% activated charcoal (Darco G60, Fluka) suspension in 1 N HCl as
described by Reinhard et al. (26). The charcoal was
sedimented, and an aliquot (100 µl) of the supernatant containing the
tritiated water was mixed with 4 ml of Ready Safe scintillant
(Beckman). Controls were obtained without BH4. To stay
under initial velocity conditions, reactions were quenched after 2 min,
and the amount of enzyme assayed was kept below 0.5 unit. One unit of
enzyme produces 1 µmol of ortho-diphenol
L-dihydroxyphenylalanine/min at 25 °C. Under these
conditions, assays were linear with time and with the amount of enzyme.
Linearity was not conserved with larger amounts of enzyme and longer
reaction times (15 min) probably because of O2 consumption.
Stocks of ferrous ammonium sulfate were stored in aliquots at
20 °C and not reused after thawing (27). Michaelis-Menten
constants were determined from Lineweaver-Burk curves analyzed by the
least squares curve fitting method with the MacCurveFit software (Kevin
Raner). To determine the pH dependence of DTH activity, the assay
buffer K-Hepes was replaced by a constant ionic strength buffer (50 mM sodium acetate, 50 mM Mes, 100 mM Tris-HCl) (28, 29). To determine the effect of dopamine
on enzyme activity, DTH was pre-incubated with dopamine for 5 min at
25 °C in the assay mixture before the reaction was started. Most
experiments were conducted in parallel for the two isoforms to compare
DTH activity and regulation in closely similar conditions. All data are
the mean of duplicate or triplicate determinations.
Phosphorylation of DTH Isoforms--
Recombinant DTH isoforms
were phosphorylated by the catalytic subunit of PKA. Either the
purified isoforms or the soluble extracts from DTH-expressing E. coli cells were used. The conditions of phosphorylation were 5-15
min at 30 °C in 25 mM Hepes, pH 7.0, 10 mM
MgCl2, 2 mM ATP, 2.5 mM spermidine,
0.5 mM EDTA, 0.5 mM EGTA, 0.025 units/µl PKA
catalytic subunit (New England Biolabs) in a volume of 25 µl.
Nonphosphorylated controls were made without PKA. TH activity was then
immediately assayed as described above. Alternatively, DTH isoforms
were phosphorylated in the presence of 0.02 mM ATP and 0.4 mCi/ml [
-32P]ATP (NEN Life Science Products). After 5 min at 30 °C, the reactions were stopped by 1 volume of SDS sample
buffer and a 2-min heating at 90 °C. Proteins were separated on 10%
SDS-PAGE, and the gels were dried and exposed for autoradiography.
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RESULTS |
Expression and Purification of Recombinant DTH Type I and Type
II--
The predicted structure of the two Drosophila
tyrosine hydroxylase isoforms is presented in Fig.
1. The positions of the regulatory and
catalytic domains are deduced from corresponding sequences in the
vertebrate TH protein (17-21). The two Drosophila enzymes only differ by the insertion of an acidic 71-amino acid segment in the
regulatory domain of DTH II.

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Fig. 1.
Structure of the Drosophila
TH protein isoforms. DTH I is a chain of 508 amino acids.
DTH II contains a specific 71-amino acid segment inserted into the
regulatory domain after the 60th residue. This additional segment
includes 17 Glu and 6 Asp and is much more acidic than the rest of the
molecule (1). The regulatory and catalytic domains indicated are
deduced from corresponding sequences in the vertebrate TH protein. The
carboxyl-terminal catalytic domain starts at residue Lys170
in DTH I and presents 61% amino acid identity with the mammalian TH
catalytic domain. The regulatory domain is not as well conserved (only
23% identity). Ser32 (asterisk) is a putative
site for cAMP-dependent protein kinase, which corresponds
to Ser40 in rat TH and human TH1. The scale indicates the
number of amino acids (AA).
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Enzymatic and regulatory properties of both DTH enzymes were analyzed
in controlled conditions after expression in E. coli BL21(DE3) cells (23) and purification. The prokaryotic expression system has been used previously to express and characterize several TH
isoforms from human and rat (21, 30-33). As an advantage, the TH
protein produced in bacteria is essentially an unphosphorylated apoenzyme that contains a very low amount of bound iron (34) and no
catecholamines. It is thus best suited to study the effect of
phosphorylation and modulators on enzymatic activity.
In cells transformed with the recombinant expression vectors pEDTHI and
pEDTHII (see "Experimental Procedures"), the addition of isopropyl
-D-thiogalactopyranoside induced an efficient
biosynthesis of DTH I and DTH II proteins, which migrated with an
apparent molecular mass of 58 and 79 kDa, respectively, on SDS-PAGE
(Fig. 2A). For DTH I, this
estimation is in agreement with the molecular mass predicted from the
coding sequence of the cDNA (57,862 Da). In contrast, the apparent
molecular mass of DTH II was higher than expected, because the sequence
predicts a protein of 65,996 Da. A minor band at 62 kDa is also
detected specifically in the DTH II-expressing cells after induction;
it is probably a degradation product. Treatment with a high salt or
alkaline pH before electrophoresis did not modify the migration of DTH
II (not shown), suggesting that this aberrant migration is not because
of protein interactions. Such an anomalous mobility in SDS gels is not
unusual with very hydrophilic proteins. The acidic segment in the
regulatory domain of DTH II may prevent regular binding of SDS
molecules and thus delay migration of the protein.

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Fig. 2.
Electrophoretic and Western blot analyses of
recombinant DTH I and DTH II proteins expressed in E. coli. A, protein pattern of BL21(DE3) cells
transformed with the plasmids pEDTHI (Type I) or pEDTHII (Type II)
before ( ) and after (+) the induction of exogenous protein
expression by isopropyl -D-thiogalactopyranoside
(IPTG). Coomassie Blue staining of total bacterial proteins
electrophoresed on an SDS-polyacrylamide gel. Migration of molecular
mass protein standards is indicated to the left of the
figure. DTH I and DTH II migrate with an apparent molecular mass of 58 and 79 kDa, respectively. A minor band at 62 kDa is also detected in
the DTH II-expressing cells after induction, which is probably a
degradation product. B, Western blot analyses of total
proteins from BL21(DE3) cells transformed with the plasmids pET-11a
(lane ø), pEDTHI (lane I), or pEDTHII
(lane II) after induction by isopropyl
-D-thiogalactopyranoside. The same blot was first
incubated with an affinity purified polyclonal anti-rat TH antibody
(Ab) (left panel) and then stripped and incubated
with a polyclonal anti-DTH II antibody (right panel).
Immunoreactive bands were revealed by chemiluminescence and visualized
by autoradiography. C, Coomassie Blue staining of
recombinant DTH I and II proteins purified as described under
"Experimental Procedures." 0.5 µg of protein of the final Mono-Q
fractions was analyzed by SDS-polyacrylamide gel electrophoresis.
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The nature of the recombinant proteins synthesized in E. coli was further checked on Western blots probed with an
affinity-purified antibody to rat TH and an antibody raised to the
specific acidic domain of DTH II. Fig. 2B (left
panel) shows that both induced peptides are recognized by the
antibody to rat TH, confirming that these molecules are TH proteins. In
addition, the antibody to DTH II recognizes, as expected, the larger
protein only (Fig. 2B, right panel). A band
migrating at the same apparent molecular weight was detected with this
antibody on a Western blot of proteins extracted from
Drosophila heads (not shown), demonstrating that the native
and recombinant DTH II proteins migrate identically on SDS-PAGE.
We found that DTH expression has to be induced at a low temperature
(18 °C) to recover soluble and active proteins. For both isoforms, a
tyrosine hydroxylase enzymatic activity was detected in the bacterial
soluble extract from the induced cells, which was strictly dependent on
the presence of iron and a reduced pteridine co-substrate in the assay
mixture. No TH enzymes (Fig. 2B, lanes ø) or TH
activity were detected in control cells transformed with the
nonrecombinant vector pET-11a.
Purification of DTH isoforms was carried out by ion exchange
fractionation and ammonium sulfate precipitation followed by Mono-Q
fractionation. The final fractions obtained were considerably enriched
(Fig. 2C), although the enzymes were not purified to homogeneity. We estimated that TH activity was enriched approximately 11-fold for DTH I and 15-fold for DTH II in the purified fractions as
compared with the bacterial soluble extracts (Table
I).
Kinetic Properties and pH Activity Profile of DTH
Isoforms--
Kinetic analysis of DTH enzymatic activity was performed
on these purified fractions. Both Drosophila TH isoforms
showed a similar temperature dependence; enzymatic activity is highest at 25-29 °C and is reduced to about 30% of the optimum at 37 °C (not shown). Therefore reactions were conducted at 25 °C, which is
the regular environmental temperature for Drosophila.
Conditions were determined (see "Experimental Procedures") in which
the rate of tyrosine hydroxylation is linear with time and enzyme
quantity (Fig. 3). In these conditions,
the specific activity of purified DTH II was found to be about twice
the specific activity of purified DTH I (Table I). The Michaelis
constants of the DTH enzymes for L-tyrosine and the
co-substrate BH4 are presented in Table
II. The two isoforms differ in their
Km value for BH4, which was found to be
reproducibly 1.5-fold lower for DTH II. BH4 has an
inhibitory effect on DTH I and II activities but at higher levels (400 µM and above) (not shown). The kinetic data obtained for
the recombinant Drosophila enzymes are comparable to the
values obtained for vertebrate TH expressed in E. coli (32,
33).

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Fig. 3.
Enzymatic activity of purified DTH
isoforms. A, L-dihydroxyphenylalanine
(L-dopa) produced as a function of time in
standard assay conditions with 1 µg of purified DTH I (open
circles) or DTH II (closed circles). The slope of the
curves is a measure of the initial velocity of the enzymatic reaction.
B, initial velocities determined as in A and
plotted for different amounts of purified DTH I (open
circles) or DTH II (closed circles). Specific enzymatic
activity can be deduced from the slope of the curves. All curves were
obtained by linear fitting with the least square method.
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Table II
Apparent Michaelis constants of recombinant Drosophila TH isoforms
Results represent the mean ± S.E. of four independent
determinations under standard conditions (pH 7 and 25 °C).
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Another significant difference found between the two DTH enzymes is
their pH activity profile (Fig. 4).
Activity of DTH I is maximal at pH 7 and much reduced at acidic and
alkaline pH. Such a bell-shaped pH profile was reported for recombinant
rat TH (35). In contrast, DTH II has a broader profile, and its activity is not markedly reduced in alkaline conditions (Fig. 4).

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Fig. 4.
pH activity profile of recombinant DTH
isoforms. Enzymatic activity of purified DTH I (open
circles) and DTH II (closed circles) was determined at
the indicated pH in the constant ionic strength buffer (see
"Experimental Procedures"). Data are the mean of three independent
experiments in which both isoforms were assayed in parallel at each pH
value. Results are expressed as percent of the mean activity observed
at pH 7 for each isoform. Bars indicate standard
errors.
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Effect of Ferrous Iron, Dopamine, and Heparin on DTH
Activity--
Vertebrate tyrosine hydroxylase is a metalloprotein that
requires ferrous iron for enzymatic activity (27, 34, 36). The iron
requirement of purified DTH I and II has been compared by varying
ferrous ion concentration in the assay mixture. We found that iron is
necessary for DTH enzymatic activation, but the iron dependence profile
is different for the two isoforms (Fig.
5). These results were confirmed with two
different enzyme preparations of each isoform. The approximate iron
concentration required for half-maximal activation of DTH I and II is
10 and 2 µM, respectively. Interestingly, DTH I activity
was not detectable at 2 µM iron, whereas a slight
activity could be detected for DTH II even with no iron added in the
assay mixture (Fig. 5). Activity was decreased at 167 µM
iron to 71 and 93% of the maximal value for DTH I and DTH II,
respectively (not shown).

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Fig. 5.
Ferrous iron requirement for the enzymatic
activity of DTH I and II. Enzymatic activity of purified DTH I
(open circles) and DTH II (closed circles) was
determined in standard conditions with various levels of ferrous
ammonium sulfate (Fe (II)) added in the assay reaction
mixture. Results are the mean of three independent experiments carried
out with two different preparations of each enzyme. Activity is
expressed as percent of the mean value determined at 55 µM ferrous iron for each DTH isoform.
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Dopamine, one of the end products of the catecholamine biosynthesis
pathway, has been shown to inhibit vertebrate TH activity (37-40). We
found that this regulation also occurs in Drosophila, although DTH seems to be less sensitive to dopamine inactivation than
vertebrate TH. Activity of both DTH isoforms is inhibited in the
presence of 20 µM dopamine, and this effect is dependent on ferrous iron concentration, the inhibition being more pronounced at
higher iron level. DTH II appears more resistant to dopamine feedback
inhibition than DTH I (Fig. 6). In
addition, lower amounts of iron are required to activate DTH II than to
allow for dopamine inhibition of its activity. Thus, at 10 µM ferrous iron, a value that could be close to the
physiological iron concentration in cells, DTH II is both fully
activated (Fig. 5) and resistant to dopamine inhibition (Fig. 6).

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Fig. 6.
Inhibition of DTH activity by dopamine.
Enzymatic activity of purified DTH isoforms was determined in the
presence or absence (control) of 20 µM dopamine with
various levels of ferrous iron in the assay mixture (6.2, 18.5, 55, and
167 µM). Activity is expressed as percent of the control
without dopamine. Open circles, DTH I; closed
circles, DTH II. Each determination is the mean of four to six
independent experiments. The ferrous iron concentration (Fe
(II)) is plotted in logarithmic scale. The asterisks
denote iron concentrations for which the effect of dopamine is
significantly different on the two DTH isoforms as determined by the
independent Student's t test (p < 0.03).
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It is known that TH activity is stimulated by the binding of polyanions
like heparin (41-43). The mammalian TH protein contains a critical
sequence for heparin binding in the regulatory domain that has been
mapped to amino acids 68-90 (44). However, this sequence is not
conserved in Drosophila TH, and we have found that 1 mg/ml
heparin in the assay mixture has no significant effect on DTH activity
(not shown).
Effect of Phosphorylation by PKA on DTH and S32R Mutant
Activity--
One of the major types of regulation of vertebrate TH is
through activation by cAMP-dependent phosphorylation of a
conserved serine residue located in the regulatory domain of the
molecule (Ser40 in rat TH) (6-8). This PKA site has been
well conserved in Drosophila TH occurring at
Ser32 (Figs. 1 and
7A). Phosphorylation of the
DTH isoforms by the catalytic subunit of PKA was carried out in the
presence of radioactive ATP. Autoradiography of the phosphorylation
product shows that DTH I and II are both rapidly phosphorylated (Fig.
7C, lanes 1 and 3). To check that the enzymes
were phosphorylated at the serine residue occurring in the conserved
PKA site, Ser32 was mutated to an arginine by site-directed
mutagenesis (Fig. 7B). The mutant isoforms thus obtained,
DTH I (S32R) and DTH II (S32R), were expressed in E. coli
and found to be enzymatically active. As shown in Fig. 7C, lanes
2 and 4, phosphorylation by PKA is abolished in these
S32R mutants.

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Fig. 7.
Phosphorylation of recombinant
Drosophila TH with PKA. A, amino acid
sequence alignment of the conserved site of phosphorylation by PKA in
the amino-terminal regulatory domain of HTH1 and DTH. The homology does
not extend further on each side of this 10-amino acid sequence.
B, sequence of the same site in the DTH(S32R) isoforms, in
which Ser32 has been mutated to an arginine by
site-directed mutagenesis. C, autoradiography of
phosphorylated proteins from bacterial soluble extracts containing
expressed DTH enzymes. 10 µg of protein of each extract was incubated
5 min at 30 °C with the catalytic subunit of PKA in the presence of
radioactive ATP. Reaction was terminated with the addition of sample
buffer followed by SDS-PAGE. Lane 1, DTH I; lane
2, DTH I (S32R); lane 3, DTH II; lane 4, DTH
II (S32R). Positions of DTH I and DTH II are indicated by the
arrows on the right side. The gel was exposed for
autoradiography for 1 h.
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Fig. 8A shows that
phosphorylation of DTH I by the catalytic subunit of PKA in the
bacterial soluble extract leads to a significant increase in TH
activity (327 ± 21%, mean of four independent experiments). The
activity of purified DTH I is also significantly stimulated by
phosphorylation with PKA (175 ± 20%, mean of three independent experiments). The activity of the mutant enzyme DTH I (S32R) is not
increased by the catalytic subunit of PKA (Fig. 8B),
demonstrating that stimulation of DTH I by PKA directly results from
the phosphorylation of Ser32.

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Fig. 8.
Effect of phosphorylation by PKA on DTH
enzymatic activity. Bacterial soluble extracts from cells
expressing the different DTH isoforms were incubated for 5 min at
30 °C with the catalytic subunit of PKA. TH activity was then
immediately assayed in standard conditions. Controls were similarly
treated but in the absence of PKA. A, DTH I; B,
DTH I (S32R); C, DTH II; D, DTH II (S32R).
Activity of the phosphorylated enzymes (gray columns) is
expressed as percent of the mean of the respective control values
(white columns). PKA significantly increases the activity of
DTH I, but not of DTH II or either mutant S32R isoforms. Results are
the mean of four independent experiments. Bars indicate
standard errors.
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In contrast to DTH I, we found that activity of the DTH II isoform is
not stimulated by PKA either in the bacterial soluble extract (Fig.
8C) or after purification (shown in Fig.
9). As expected, the mutant enzyme DTH II
(S32R) is not regulated by PKA as well (Fig. 8D). However,
phosphorylation of DTH II by PKA activates the enzyme in the presence
of dopamine and a high iron concentration, bringing back the enzyme
activity close to the level observed in the absence of dopamine (Fig.
9). The phosphorylated enzyme even appears slightly more active in the
presence of dopamine (Fig. 9), probably because dopamine binding
stabilizes TH (39). Thus, phosphorylation by PKA has no effect on basal
DTH II activity and only activates efficiently the enzyme inhibited by
dopamine.

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Fig. 9.
Phosphorylation of DTH II antagonizes
dopamine inhibition. Purified DTH II was incubated for 15 min at
30 °C with (+) or without ( ) the catalytic
subunit of PKA and then immediately assayed in the presence
(+) or absence ( ) of 20 µM
dopamine. The assay mixture contained 167 µM ferrous
ammonium sulfate to increase the inhibitory effect of dopamine on
enzymatic activity (see Fig. 6). Phosphorylation activates the enzyme
only in the presence of dopamine. Results are expressed as percent of
the activity of the unphosphorylated enzyme assayed in the absence of
dopamine. Bars indicate the standard errors of triplicate
determinations. The data significantly different from the control
values are denoted by asterisks. *, p < 0.05; **, p < 0.005 as determined by independent
Student's t test.
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DISCUSSION |
Alternative splicing of the TH primary transcript generates two
different TH isoforms in Drosophila. The major isoform, DTH II, contains a specific 71-amino acid hydrophilic segment in the amino-terminal regulatory domain (Fig. 1). Messenger RNA localization showed that DTH I is only expressed in the central nervous system, and
DTH II is expressed at a high level in the epidermis (1). In addition,
no DTH II protein can be detected in the Drosophila central
nervous system by immunolabeling with a polyclonal antibody to the
specific hydrophilic segment of DTH
II.2 Alternative splicing of
Drosophila TH is then strictly tissue-specific. To address
the physiological relevance of these observations, it was interesting
to compare the enzymatic and regulatory properties of the DTH isoforms.
Because of the low level of TH activity in Drosophila, it
would have been very difficult to extract and purify the TH enzymes
from insect tissues. Therefore, the two isoforms were analyzed after
high level expression in E. coli and purification. In
addition, because catecholamine biosynthesis or
cAMP-dependent phosphorylation do not occur in bacteria,
the prokaryotic expression system is well adapted to study TH regulation.
The Drosophila TH isoforms expressed in E. coli
were found to be enzymatically active. In addition, they are bound by
TH-specific antibodies (Fig. 2). The anomalous migration of DTH II on
SDS-PAGE indicates that the additional hydrophilic segment may alter
the structure of the protein or prevent regular binding of SDS
molecules. Although purified DTH II seems to have a higher specific
activity than purified DTH I, the enzymes were probably not pure enough to allow for precise determination of their catalytic constants. In
addition, our results show that activity of the DTH enzymes dramatically depends on assay conditions.
The Km for tyrosine was found to be comparable for
the two DTH isoforms. In contrast, the Km value for
BH4 is moderately but significantly lower for DTH II (Table
II). Thus, the presence of an additional acidic amino acid segment in
the regulatory domain of DTH II decreases the Km for
BH4 as compared with DTH I. Interestingly, it has been
reported that phosphorylation of recombinant rat TH by PKA decreases
the Km value for BH4 (32) (see below).
TH is a metalloprotein that requires ferrous iron for activity (27, 34,
36). A site-directed mutagenesis study (45) and crystal structure of
the catalytic domain of rat TH (46) have shown that the iron atom is
localized within the active site cleft of the enzyme and is coordinated
by three amino acid residues: His-331, His-336, and Glu-376. These
amino acids are conserved in the Drosophila enzymes,
corresponding to His-338, His-343, and Glu-383, respectively, in DTH I. Native TH purified from vertebrate tissues contain bound
catecholamines, and catecholamine binding prevents the release of the
bound iron (47-49). Because E. coli contains no
catecholamines, iron may be less strongly bound to TH enzymes expressed
in bacteria than to native TH enzymes extracted from tissues. When
expressed in E. coli and purified, the human TH isoforms are
indeed recovered as practically iron-free apoenzymes (32, 34). In
addition, the purification buffer we used contains dithiothreitol,
which has been shown to increase the rate of iron release from TH (34).
Consequently, the purified DTH isoforms can be similarly considered as
apoenzymes practically devoid of iron.
Although the two Drosophila TH enzymes have been purified by
the same procedure, our data show that DTH I and DTH II require different levels of iron to be activated (Fig. 5). Very low levels of
iron are sufficient to ensure full activation of DTH II. The following
two hypotheses can be proposed to interpret this observation: either
the iron binding site is less accessible in DTH I than in DTH II or the
regulatory acidic domain in DTH II increases enzyme affinity for iron.
Consequently, we could predict that the central nervous system enzyme
DTH I would be more sensitive than DTH II to cellular variations in the
level of iron. Could iron be an endogenous regulator of TH activity?
Free iron in the cytosol is kept at a low level (probably below 10 µM) by incorporation into protein chelators like ferritin
(50). Human brain TH extracted from the caudate nucleus is activated
more than 10-fold by incubation with ferrous ion (51), and Haavik
et al. (34) have proposed that a significant fraction of
human TH is not saturated with iron in vivo. In addition,
these authors have reported that recombinant HTH1 requires a lower
level of iron (7.8 µM) than HTH2 (13.5 µM) for half-maximal activation. These results and ours suggest that it
would be interesting to compare the iron activity profile for the
different vertebrate TH isoforms.
Dopamine and other catecholamines inhibit and stabilize vertebrate TH
activity (37-40), and iron stimulates dopamine binding (31). We have
shown here that this regulation is conserved in Drosophila
TH and that the level of inhibition by dopamine depends on ferrous iron
concentration. For vertebrate TH, it has been demonstrated that
catecholamine binds to the active site iron oxidized in a ferric redox
state (47, 49), thus stabilizing the enzyme in an inactivated form
(32). Phosphorylation by PKA decreases the binding affinity of human TH
for dopamine (40, 52), and conformation studies suggest that the
catalytic site lays in close proximity or interacts with the PKA
phosphorylation site of the regulatory domain (53). Deletion of the
NH2-terminal regulatory domain of human TH1 up to the PKA
phosphorylation site (Ser40) abolishes the inhibitory
effect of dopamine (54). All these data strongly suggest that: 1) there
is a direct interaction between the region surrounding the PKA site in
the regulatory domain, and the iron binding site occurring close to the
active site in the catalytic domain; 2) this interaction is required
for dopamine binding and inhibition of TH activity; and 3) this
interaction is relieved by phosphorylation of Ser40.
Interestingly, DTH II seems to be more resistant than DTH I to dopamine
inhibition (Fig. 6), suggesting again that this isoform is at least
partially in an activated state comparable to that of
phosphorylated TH.
Another major difference we observed between DTH I and DTH II is the
effect of phosphorylation. Both TH isoforms are good substrates for the
PKA catalytic subunit (Fig. 7). Site-directed mutagenesis of
Ser32 to an arginine abolished phosphorylation,
demonstrating that only Ser32 can be phosphorylated by PKA
in Drosophila TH. Similar results have been obtained in
previous studies by site-directed mutagenesis of the homologous
Ser40 in rat TH (32, 55). We have found that
phosphorylation by PKA markedly stimulates DTH I activity in the
absence of catecholamine. For recombinant rat TH expressed in E. coli, it has been shown that phosphorylation by PKA in the absence
of dopamine results in a moderate decrease in the Km
for BH4 and no increase in Vmax
(32). Such a decrease in Km was shown to induce only
an apparent 2-fold activation of the human isoform HTH1 when assayed in
standard initial velocity conditions (33). Therefore, a decrease in
Km is probably not sufficient to explain the strong
activation of DTH1 by PKA phosphorylation we observed in the bacterial
extract in the absence of dopamine (Fig. 8). We have preliminary data
suggesting that phosphorylation of DTH I by PKA increases the
Vmax for BH4 in these conditions
(not shown), as is the case for recombinant rat TH in the presence of
dopamine (32). Further work is needed to clarify this apparent
discrepancy between mammalian TH and Drosophila TH I.
In contrast to DTH I, phosphorylation by PKA does not regulate DTH II
in the absence of dopamine. It has been shown that phosphorylation of
HTH1 by PKA induces a structural transition in the enzyme (53). Because
DTH II is readily phosphorylated by PKA (Fig. 7), two hypothesis could
be proposed for the lack of effect of phosphorylation on DTH II
activity in the absence of dopamine. Either the amino acid segment
inserted in the regulatory region prevents a conformational change
required for enzyme activation or alternatively the TH enzyme
containing this segment spontaneously presents a conformation similar
to that of the phosphorylated enzyme. A comparable lack of regulation
by PKA has been recently reported for the human isoform HTH3 in the
absence of dopamine (33). On the opposite side, we show that
phosphorylation of DTH II by PKA activates the enzyme in the presence
of dopamine (Fig. 9). This result is in agreement with the proposal
that the major mode of activation of TH by phosphorylation is the
alleviation of catecholamine inhibition (31, 32, 56). We propose that
the structure of the regulatory domain in DTH II precludes activation
by phosphorylation in the absence of dopamine because the enzyme is
already in a partially activated conformation, but phosphorylation can
still reactivate the enzyme inhibited by dopamine. In the insect
epidermis where DTH II is expressed, the unique properties of this
isoform could be an advantage because a large amount of catecholamines
has to be produced in a short period of time when a new cuticle is
synthesized. In the central nervous system a precise regulation
of dopamine biosynthesis is required, and it is probably useful for
synapse plasticity that the nervous system-specific isoform DTH I can be rapidly stimulated by cAMP-dependent phosphorylation
even before catecholamines have accumulated.
Finally, the pH dependence also differs significantly for the two
Drosophila TH enzymes. DTH II has a broader pH profile and is more active than DTH I at basic pH (Fig. 4). Interestingly, it is
known that regulation of mammalian TH changes the pH dependence of the
enzyme; activation by cAMP-dependent phosphorylation
broadens the pH profile and shifts the pH optimum toward the basic side (8, 57), and inhibition by dopamine shifts the pH activity profile
toward the acidic side (31, 54). Clearly, the pH dependence of DTH II
resembles the profile of an activated TH enzyme. In addition,
steady-state intracellular pH is 7.4-7.6 in Drosophila (58,
59). This slightly alkaline value is even increased in the presence of
20-hydroxyecdysone (+ 0.3 units) (59), a hormone that plays an
important role in the larval moulting process. Therefore, the pH
activity profile of DTH II could be an advantage for rapid dopamine
biosynthesis in the epidermis during moulting.
In conclusion, we have shown in this study that a structural difference
in the regulatory domain of Drosophila TH isoforms affects
several properties of the enzymes, such as the Km for BH4, pH dependence, ferrous iron activation, regulation
by dopamine, and cAMP-dependent phosphorylation. Several
lines of evidence indicate that the acidic segment in the regulatory
domain of DTH II stabilizes the enzyme in an activated conformation. Conservation of a functional Ser40 PKA site in all the TH
sequences through evolution argues that this site is an essential
feature of the enzyme. In vivo genetic experiments are now
in progress to characterize further the role of the two DTH isoforms
and the function of this PKA site in tissue-specific regulation of
dopamine biosynthesis in Drosophila.