Mutation of Tyrosine 332 to Phenylalanine Converts
Dopa Decarboxylase into a Decarboxylation-dependent
Oxidative Deaminase*
Mariarita
Bertoldi,
Marco
Gonsalvi,
Roberto
Contestabile
, and
Carla Borri
Voltattorni§
From the Dipartimento di Scienze Neurologiche e della Visione,
Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia,
Università degli Studi di Verona, Strada Le Grazie, 8, 37134 Verona, Italy, and the Dipartimento di Scienze
Biochimiche "A. Rossi Fanelli" and Centro di Biologia Molecolare
del Consiglio Nazionale delle Ricerche, Università "La
Sapienza," 00185 Roma, Italy
Received for publication, May 17, 2002, and in revised form, July 11, 2002
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ABSTRACT |
A flexible loop (residues 328-339), presumably
covering the active site upon substrate binding, has been
revealed in 3,4-dihydroxyphenylalanine decarboxylase by means of
kinetic and structural studies. The function of tyrosine 332 has been
investigated by substituting it with phenylalanine. Y332F displays
coenzyme content and spectroscopic features identical to those of the
wild type. Unlike wild type, during reactions with
L-aromatic amino acids under both aerobic and
anaerobic conditions, Y332F does not catalyze the formation of aromatic
amines. However, analysis of the products shows that in aerobiosis,
L-aromatic amino acids are converted into the corresponding aromatic aldehydes, ammonia, and CO2 with concomitant
O2 consumption. Therefore, substitution of Tyr-332 with
phenylalanine results in the suppression of the original activity and
in the generation of a decarboxylation-dependent oxidative
deaminase activity. In anaerobiosis, Y332F catalyzes exclusively a
decarboxylation-dependent transamination of
L-aromatic amino acids. A role of Tyr-332 in the C
protonation step that catalyzes the formation of physiological products
has been proposed. Furthermore, Y332F catalyzes oxidative deamination
of aromatic amines and half-transamination of D-aromatic amino acids with kcat values comparable with
those of the wild type. However, for all the mutant-catalyzed
reactions, an increase in Km values is observed,
suggesting that Y 
F replacement also affects substrate binding.
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INTRODUCTION |
Dopa1 decarboxylase
(DDC; EC 4.1.1.28) is a homodimeric pyridoxal 5'-phosphate (PLP) enzyme
that catalyzes as the main reaction the decarboxylation of
L-aromatic amino acids into the corresponding aromatic
amines shown in Reaction 1.
Side reactions with turnover times measured in minutes are also
catalyzed by the enzyme. In particular, DDC exhibits half-transaminase activity toward D-aromatic amino acids (1) and oxidative
deaminase activity toward aromatic amines (2, 3), as shown in Reactions 2 and 3.
Studies on the effect exerted by O2 on reaction
specificity of the enzyme have shown that under anaerobic conditions,
Reaction 1 takes place with a kcat value
approximately half that occurring in the presence of O2 and
is accompanied by a decarboxylation-dependent transamination
(4), and Reaction 2 occurs at the same extent either in the presence or
absence of O2 (4). Reaction 3 does not occur in
anaerobiosis and is replaced by half-transamination (1, 5).
Tancini et al. (6) reported the presence of an exposed and
flexible region in the native pig kidney DDC molecule susceptible to
tryptic digestion by which two fragments cleaved at the
Lys-334-His-335 bond were formed. Although the nicked enzymatic
species does not exhibit either decarboxylase or oxidative deamination
activities, it retains a large percentage of the native transaminase
activity toward D-aromatic amino acids and displays a slow
transaminase activity toward aromatic amines (1). Steady-state kinetic
studies of native and nicked enzymatic species together with protection experiments against limited proteolysis of DDC by various substrates have suggested that ligand conformational changes occur at or near the
tryptic cleavage region (1). The finding that recombinant rat liver DDC
lacking this loop is incompetent for decarboxylation supports this view
(7).
The spatial structure of ligand-free DDC and its complex with the
anti-Parkinson drug carbiDopa has been recently solved, but the
flexible loop between residues 328 and 339 is invisible in the electron
density map (8). A model based on the coordinates of the enzyme with
the flexible loop in its hypothetical open form was built. Although in
this modeled structure the loop is located at the dimer interface, far
away from the active site, it is expected to extend toward the active
site of the other monomer in a closed conformation upon substrate
binding. Such loop closure could be an essential step in the catalytic
mechanism of the enzyme, and it is also reasonable to suppose that some
loop residues could even take part in the catalytic mechanism. Fig.
1 shows a tentative model of the flexible
loop in a position to gate access to the active site in the
DDC-carbiDopa complex. The tyrosyl residue, Tyr-332, in the model
is found 3.86 Å from the -carbon of the ligand. This
residue is of particular interest as it is conserved in
PLP-dependent group II -decarboxylases (9).
Site-directed mutagenesis has been used to investigate the role of
Tyr-332 in the mobile loop of DDC.
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Fig. 1.
Active site view of the
energy-minimized model zooming in on Tyr-332 of the flexible loop of
DDC in complex with carbiDopa. Several residues around the
-carbon of the ligand, which is depicted in red, are
represented in wire-frame mode. PLP is colored in
yellow. Tyr-332, depicted in ball-and stick mode,
and residues colored in gray belong to the neighboring
subunit. The flexible loop was modeled using the coordinates 1JS3
deposited in the Protein Data Bank (8). Energy computations were done
with the GROMOS96 (27) implementation of Swiss-Pdb Viewer (28).
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EXPERIMENTAL PROCEDURES |
Chemicals--
L-Dopa, L- and
D-5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT,
serotonin), dopamine, PLP, pyridoxamine 5'-phosphate (PMP), NADH,
bovine liver L-glutamate dehydrogenase, horse liver alcohol
dehydrogenase, and Hepes were Sigma products. The liquid chromatography solvents (HPLC grade) were from Labscan. Ingredients for
bacterial growth were from Difco. Oligonucleotides were from Invitrogen. PCR amplifications were performed using the Expand high
fidelity PCR system commercialized by Stratagene. The restriction enzymes used for cloning were from Biolabs. D,
L-[1-14C]Dopa (55 mCi/mmol) was a product of
ICN Pharmaceuticals. All other chemicals were of the highest purity available.
Site-directed Mutagenesis--
Site-directed mutagenesis was
performed by overlap extension PCR (10). This method uses four
oligonucleotide primers in three separate PCR reactions to introduce a
mutation into the target DNA sequence. Two separate PCR reactions are
run, one using primers 1 and 3 to amplify a portion of the target
sequence and the other using primers 2 and 4 to amplify the other
portion. A short section of DNA has identical sequence in both PCR
products and corresponds to the sequence of primers 2 and 3 ("internal primers"), which are complementary to each other and
carry the "mismatch," i.e. the mutation. In the third
PCR, the purified products of the previous two reactions are employed
as template. Following denaturation, a small fraction of the template
DNA will anneal to form heteroduplexes and will be extended at its
recessed 3' ends by the polymerase used in the reaction. The
full-length sequence containing the mutation is then amplified using
primers 1 and 4 ("external primers"). This mutant was
produced using as external primers 5'-ATCGGCTCGTATAATGTGTGG-3' and
5'-GTTCTGATTTAATCTGTATCAGG-3'. Internal primers were
5'-GACCCCGTGTTCTTAAAGCAC-3' and
5'-GTGCTTTAAGAACACGGGGTC-3'. The newly inserted part
of the expression construct, pKKDDC-mutant, was sequenced to confirm
mutation, and the plasmid was used to transform Escherichia coli
SVS370.
Expression and Purification of Y332F Mutant--
The conditions
used for expression and purification of the mutant protein in E. coli (SVS370) were as described for the wild-type enzyme (2, 11).
Since the mutant enzyme does not show detectable decarboxylase activity
in the standard spectrophotometric assay, which measures production of
aromatic amines, screening with antibodies to native DDC was therefore
necessary to monitor the purification procedure. The purified mutant
was homogenous as indicated by a single band on SDS-PAGE. The enzyme
concentration was determined by using an 
M of 1.3 × 105M
1cm1. PLP
content of holoDDC enzymes was determined by releasing the coenzyme in
0.1 M NaOH and by using M = 6600 M1 cm1 at 388 nm.
Western Blotting--
A sample of 20 µg of protein was
subjected to SDS-polyacrylamide gel electrophoresis using a 12.5%
acrylamide gel. The proteins were electroblotted to
Immobilon-P-membranes (Millipore), and Western blot analysis was
performed according to Gallagher et al. (12).
Enzyme Assays--
DDC mutant Y332F (2-5 µM) was
incubated with (0.1-5 mM) L-Dopa or
L-5-HTP in 50 mM Hepes, pH 7.5, at 25 °C in
the presence or absence of O2. Production of dopamine or
5-HT was determined with a spectrophotometric assay outlined by Sherald
et al. (13) and modified by Charteris and John (14).
Alternatively, production of aromatic amines as well as consumption of
L-aromatic amino acids were measured by HPLC analysis.
Aliquots were removed at time intervals, and trichloroacetic acid was
added to a final concentration of 5% (v/v). The quenched solutions
were centrifuged to remove protein, and the supernatants were analyzed
using a Discovery (Supelco) C18 column (4.6 × 250 mm). The eluent
was methanol:acetic acid:H2O, 24:1:75 with 6 mM
octanesulfonic acid at a flow rate of 0.6 ml/min. Detection was set at
280 nm. The concentration of L-aromatic amino acids and
aromatic amines in the analyzed samples was determined from a standard
curve generated from known concentrations of the compounds with respect
to the internal standard. The amounts of ammonia and aromatic aldehyde (produced during the reaction of the Y332F mutant with
L-aromatic amino acids or aromatic amines) were determined
using the coupled assays with glutamate dehydrogenase and alcohol
dehydrogenase, respectively, as described (2). The amount of aldehyde
or ammonia was measured by the decrease in absorbance at 340 nm due to
the conversion of NADH to NAD+. The rate of production of
14CO2 during the reaction of mutant with
[1-14C]Dopa was determined as described (4).
Radioactivity was determined with a Beckman Instruments LS 1801 liquid
scintillation counter. H2O2 production and
O2 consumption were measured according to Refs 1 and
3, respectively. The detection and quantification of PLP and PMP
content were performed using the HPLC procedure described previously
(1, 4). The apparent pseudo first-order rate constants,
kobs, of the reaction of the Y332F mutant at
varying concentrations of D-5-HTP were obtained by
measuring the decrease of the 425-nm absorbance band as described (1).
The dependence of kobs on D-5HTP
concentrations exhibits a saturation behavior, and a hyperbolic fit
gives the value of apparent dissociation constant,
KD, and the maximum value of rate constant, kmax. Enzymic assays were performed under anaerobic
conditions with 1 ml of Reacti-Vials (Aldrich) as described previously
(3, 4).
Spectral Measurements--
Absorption spectra were recorded in a
Jasco V-550 spectrophotometer. CD measurements were carried out
in a Jasco J-710 spectropolarimeter at a scan speed of 50 nm/min with a
bandwidth of 2 nm.
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RESULTS |
Reaction of Y332F Mutant with L-Aromatic Amino Acids
under Aerobic and Anaerobic Conditions--
Like the wild type, the
mutant binds 2 mol of PLP per dimer. Absorption and CD spectra of the
Y332F mutant in the UV-visible and far UV region are essentially
identical to those of the wild-type enzyme (data not shown). When the
Y332F mutant was incubated at 25 °C under aerobic conditions with
L-Dopa, no dopamine formation was detected either by the
spectrophotometric or the HPLC assays. However, L-Dopa
level decreases, and its decrease parallels the production of
3,4-dihydroxyphenylacetaldehyde and ammonia accompanied by
O2 consumption in a 1:2 molar ratio with respect to the
products (Fig. 2). When the reaction was
performed under the same experimental conditions in the presence of
[1-14C]Dopa, 14CO2 release was
observed. The initial velocities of these catalytic events are reported
in Table I. Likewise, although no 5-HT
production could be detected during the reaction of Y332F with
L-5-HTP, conversion of L-5HTP into
5-hydroxyindolacetaldehyde and ammonia as well as consumption of
O2 in a 1:2 molar ratio with respect to the products take
place with the initial velocity values reported in Table I. During the
reaction of Y332F, either with L-Dopa or with
L-5-HTP, no detectable H2O2 was
found. Initial velocities of oxidase activity, measured as
L-aromatic amino acid consumption at varying concentrations
of L-Dopa or L-5-HTP, have been determined. The
kcat and Km values are
reported in Table II. As for the wild
type, upon addition of L-Dopa to Y332F, an increased absorption centered at 425 nm immediately appears. This absorbance band, attributed to the external aldimine, decreases with time, and
after the time required for consumption of substrate, the original
425-nm absorbance of the holoenzyme reappears (Fig.
3). Qualitatively identical spectral
changes are observed upon addition of L-5-HTP to the
mutant. When the reaction of Y332F with L-Dopa or
L-5-HTP was performed under anaerobic conditions, the
concentration of the substrates remained almost unchanged with time,
and neither dopamine nor 5-HT were produced. Instead, a conversion of
PLP into PMP takes place very quickly: at 30 s, 89% (for
L-Dopa) and 55% (for L-5-HTP) of the original
coenzyme content are transformed into PMP.
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Fig. 2.
Consumption of L-Dopa and
O2 and formation of ammonia and 3, 4-dihydroxyphenylacetaldehyde during the reaction of Y332F mutant with
L-Dopa. Y332F (2.4 µM) was incubated in
50 mM Hepes, pH 7.5, with 200 µM
L-Dopa. For L-Dopa determination, 150-µl
samples were withdrawn at the indicated times and denatured with
trichloroacetic acid. After removal of the precipitated protein by
centrifugation, the supernatants were subjected to HPLC, and the areas
of the peaks were converted to absolute to as described under
"Experimental Procedures"):  , L-Dopa;  , 3, 4-dihydroxyphenylacetaldehyde;  , ammonia, measured by the coupled
alcohol and glutamate dehydrogenase systems, respectively;  ,
O2 consumed measured by a Clark oxygen electrode. Data
shown are means of three independent experiments. Error bars
denote standard errors from mean values. Dotted lines
were theoretical from a fit to the integrated form of the following
equations:  d[L-Dopa] = d[product] = k [L-Dopa]
dt for L-Dopa consumption and products
formation, respectively. Straight lines were theoretical
from a linear regression fit during the initial linear phase.
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Table I
Initial velocities of reaction of Y332F mutant (2.4 µM)
with 200 µM L-Dopa or L-5-HTP
Numbers represent initial velocities expressed as mol/sec/mol enzyme.
Data shown are means of three independent experiments; S.E. mean in
each case was less than 6% of the mean value.
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Table II
Apparent kinetic properties for wild-type DDC and Y332F mutant
Oxidative deaminase activity was determined by measuring either
production of aromatic aldehyde or ammonia or consumption of
L-aromatic amino acids (see Experimental Procedures).
Half-transaminase activity was determined by measuring the rate
constants of the 425-nm absorbance decrease at varying D-5-HTP
concentrations, as described under "Experimental Procedures."
Decarboxylase activity was determined by measuring production of
aromatic amines, as described under "Experimental Procedures."
ND, not detected. kcat and Km
were obtained from nonlinear regression fit to the Michaelis-Menten
equation using SigmaPlot2000 (SPSS). The errors reported are the
standard error values derived by the curve fitting program.
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Fig. 3.
Time-dependent
absorbance changes occurring upon addition of
L-Dopa to Y332F mutant. The enzyme (2.9 µM) (dashed line) in 50 mM Hepes,
pH 7.5, was treated with L-Dopa (200 µM), and
spectra were recorded at the indicated times.
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Reaction of Mutant with Aromatic Amines and D-Aromatic
Amino Acids--
Despite its total impairment of aromatic amines
generation, the Y332F mutant retains substantial catalytic competence
for the oxidative deamination of aromatic amines. In fact, reaction of
the Y332F mutant with 5-HT or dopamine produces the corresponding aromatic aldehyde (5-hydroxyindolacetaldehyde or 3, 4-dihydroxyphenylacetaldehyde) and ammonia in equivalent amounts and
consumes O2. On the other hand, aromatic amines undergo
half-transamination under anaerobic conditions with rate constants
similar to those of wild type (< 0.1 min1) (1, 5).
Reaction of D-5-HTP with the mutant results in time-dependent spectral changes consisting of a decrease of
the 425-nm absorbance band and a concomitant increase in the 330-nm region. These spectral changes correspond to conversion of PLP-bound to
PMP (data not shown). This behavior is identical to that already observed with wild type (1-3). Steady-state kinetic parameters were
determined for both oxidative deaminase and half-transaminase activities and reported in Table II. To allow a comparison, the values
of the wild-type DDC are also included. The Y332F mutant shows
negligible changes from the wild type in the
kcat value for oxidative deamination and
in the kmax of half-transamination but has
increased Km values (for aromatic amines) and increased KD values (for D-5-HTP) as
compared with those of the wild-type enzyme.
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DISCUSSION |
Flexible loops that occlude active sites during catalysis are
recognized as structural elements common to many enzymes. Loop closure
induced by substrate binding has great importance in catalysis and
specificity by recruiting functional groups into the active site
(15-17), stabilizing reactive intermediates (18-20), and preventing the formation of stable abortive complexes (21). Kinetic and structural
data have indicated that an 11-residue loop in DDC is conformationally
dynamic, suggesting that this loop closes over the active site after
substrate binding. This study was prompted to gain insights into the
function of the mobile residue of this loop, Tyr-332.
Replacement of Tyr-332 with phenylalanine results in a protein that is
still capable of selectively cleaving the correct bond between C and
COOH of L-aromatic amino acids. This is supported by the
finding that during the reaction of the mutant with L-Dopa in aerobiosis, CO2 is released, and by the occurrence in
anaerobiosis of a decarboxylation-dependent transamination
of both L-Dopa and L-5-HTP. In fact, PMP
formation would be promoted along the reaction pathway if the
decarboxylated substrate (quinoid) intermediate is protonated at C4'
instead at C. On the basis of these data, it can be anticipated that
DDC does not require Tyr-332 for the decarboxylation step.
Nevertheless, the Y332F mutant is unable to generate aromatic amines to
any discernible extent. Since reprotonation at C after
decarboxylation is necessary for the generation of aromatic amines
(Scheme 1 (a)), a role for
Tyr-332 residue as a proton donor in this reprotonation step can be
advanced. However, no accumulation of quinonoid intermediate during the reaction of the Y332F mutant with L-aromatic amino acids
can be observed. This would be explained by the fact that, although the original overall decarboxylation of the wild-type enzyme is completely suppressed, a new catalytic activity is generated in the presence of
O2. It consists of a decarboxylation-dependent
oxidative deamination converting L-aromatic amino acids
into CO2, aromatic aldehydes, and ammonia occurring with a
consumption of molecular oxygen in a 1:2 molar ratio with respect to
the products. An oxidase activity displaying the same stoichiometry has
been already reported to be catalyzed by the wild-type enzyme
toward aromatic amines (2, 3). Analogous with the already proposed
mechanism for the latter reaction (5), it is reasonable to postulate
that decarboxylation-dependent oxidative deamination of
L-aromatic amino acids catalyzed by the Y332F mutant occurs
according to the mechanism outlined in Scheme 1 (b). After
release of CO2, binding of O2 to the C4' of the
quinonoid intermediate could lead to the formation of a peroxide anion
that, once stabilized by protonation to the hydroperoxyPLP intermediate, will undergo heterolysis of the O-O bond. This allows regeneration of PLP and formation of an imine complex that
spontaneously decomposes to aromatic aldehyde and ammonia. According to
this mechanism, reaction of Y332F with L-aromatic amino
acids does not occur through a serial mechanism in which aromatic
amine, produced by decarboxylation, is a transient intermediate that is
subsequently converted into aldehyde and ammonia. This is consistent with our data. In fact, the kcat values for
oxidative deamination of aromatic amines catalyzed by the Y332F mutant,
similar to those catalyzed by the wild-type DDC, are about 150-fold
lower than those catalyzed by the mutant toward the corresponding
L-aromatic amino acids (Table II). Moreover, reaction of
the Y332F mutant with L-aromatic amino acids under
anaerobic conditions is characterized by no accumulation of aromatic
amine. In an O2-free atmosphere, if the amine were
produced, it would accumulate since it would undergo a very slow
transamination.
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Scheme 1.
Proposed pathways for
reactions of Y332F mutant with L-aromatic amino acids
(b) in the presence or (c) in the
absence of O2; (a) represents the pathway
for the overall decarboxylation.
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ABSTRACT
INTRODUCTION
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