Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme

Article abstract of DOI:10.1016/j.bbrc.2011.12.124

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46. mM) and dopa (km 1.40. mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine.

Full text of DOI:10.1016/j.bbrc.2011.12.124

Biochemical and Biophysical Research Communications 418 (2012) 211–216
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel
4-hydroxyphenylacetaldehyde synthase enzyme
Michael P. Torrens-Spence a, Glenda Gillaspy a, Bingyu Zhao b, Kim Harich a, Robert H. White a,
Jianyong Li a,
⇑
a Department of Biochemistry, Virginia Tech, Blacksburg, VA, United States
b Department of Horticulture, Virginia Tech, Blacksburg, VA, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 November 2011
Available online 11 January 2012
Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic
acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several
characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this
limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized
plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was dem-
onstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a
unique substrate specificity towards tyrosine (km 0.46 mM) and dopa (km 1.40 mM). Metabolite analysis
established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates
that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this
gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first
reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single
enzyme capable of producing 4-HPAA from tyrosine.
Keywords:
Aromatic amino acid decarboxylases
Aromatic acetaldehyde syntheses
Tyrosine decarboxylase
4-Hydroxyphenylacetaldehyde
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
name provides misleading functional implications. This issue with
ambiguous AAAD annotation also applies to plants. Database and
AAAD is an eponym originally applied to mammalian dopa
decarboxylase (DDC). This enzyme catalyzes the respective decar-
boxylation of dopa and 5-hydroxytryptophan to dopamine and 5-
hydroxytryptamine. The AAAD designation has subsequently been
used for annotating similar enzymes from many other species.
Such annotations are appropriate for species that have a single
AAAD protein with well-defined substrate specificity. This same
annotation is vague or inaccurate for organisms with multiple
divergent AAAD-like proteins. For example, an expansion of the
AAAD gene within insect and plant species results in a multiplicity
of functionally diverse AAAD enzymes. A single AAAD annotation
does not accurately represent the selection of activities and sub-
strate specificities. This can be illustrated through our recent study
of some predicted Drosophila and mosquito AAAD sequences.
Resulting research determined that these AAAD like sequences cat-
alyze a complicated decarboxylation–deamination process of dopa
to 3,4-dihydroxy-phenylacetaldehyde (DHPA) [1]. These enzymes,
unlike their names imply, have nothing to do with the production
of arylalkylamines, but rather catalyze the production of their cor-
responding aromatic acetaldehydes. Consequently, their AAAD
literature searches reveal that plants have similarly diverse AAAD
groups. For example, one particular plant AAAD group catalyzes
the same decarboxylation–deamination process as the aforemen-
tioned insect AAAD proteins.
Despite the major difference in catalytic processes between
amine and aldehyde producing AAADs, this enzyme family in gen-
eral retains great homology. This sequence similarity makes for
problematic primary sequence differentiation of the amine produc-
ing AAADs and aldehyde producing AASs. In particular, this large
level of homology makes it difficult to distinguish plant AASs from
plant tyrosine decarboxylases (TyDCs). To investigate an alternate
AAAD differentiation approach, we have been investigating the
spectral characteristics of various AAAD enzymes. During this anal-
ysis, we observed no consistent spectral pattern for the differenti-
ation of AAADs and AASs. This investigation did however enable
characterizations of several previously investigated plant AAADs
and AASs. Upon completion, we determined that contrary to previ-
ous reporting [2], parsley (Petroselinum crispum) enzyme (NCBI
accession Q06086) is not capable of catalyzing the conversion of
tyrosine to tyramine and dopa to dopamine, but rather catalyzes
the conversion of tyrosine to 4-HPAA and dopa to DHPA. This
substrate specificity for tyrosine and dopa makes parsley Q06086
unique amongst all other previously characterized plant AASs. To
clearly differentiate this novel AAS activity from a typical AAAD
⇑
Corresponding author.
E-mail address: lij@vt.edu (J. Li).
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2011.12.124

212
M.P. Torrens-Spence et al. / Biochemical and Biophysical Research Communications 418 (2012) 211–216
we performed a side-by-side comparison of this parsley AAS en-
zyme to a highly homologous (70% identity) Thalictrum flavum
TyDC (NCBI accession AAG60665). We also provide data showing
that tyrosine is the sole physiological substrate for Q06086. The
resulting information reveals the first plant AAS with tyrosine as
a substrate. Q06086 and AAG60665 also serve as great examples
illustrating that AAAD-like proteins sharing high sequence homol-
ogy may have completely different biochemical functions.
phosphate buffer (pH 6.8) and incubated at 25 °C in a water bath.
At different time periods after incubation (usually 10–60 min)
the reaction of individual reaction mixtures was stopped by mixing
an equal volume of 0.8 M formic acid. Supernatants of the reaction
mixtures, obtained by centrifugation, were analyzed by HPLC with
electrochemical detection (ED) as previously described [1]. Some
reaction mixtures were treated with an equal volume of NaBH4 sat-
urated ethanol, incubated for 5 min at 25 °C, treated with 0.8 M
formic acid (decompose remaining NaBH4), and then analyzed by
HPLC-ED similar to those described in a previous report [1]. The
mobile phase consisted of 50 mM monopotassium phosphate (pH
4.6) containing 13% (v/v) acetonitrile and 0.5 mM octyl sulfate.
2. Materials and methods
2.1. Reagents
2.6. GC/MS analysis of parsley Q06086-catalyzed product from
tyrosine
Tyrosine, tyramine, dopa, dopamine, benzaldehyde, 4-hydroxy-
benaldehyde, 3,4-dihydroxybenaldehyde, 4-hydroxyphenyletha-
nol, 3,4-dihydroxyphenylethanol, pyridoxal 5-phsophate (PLP),
formic acid, and acetonitrile were purchased from Sigma (St. Louis,
MO). The IMPACT-CN protein expression system was purchased
from New England Biolabs (Ipswich, MA).
Enzyme activity assay strongly indicated the production of 4-
HPAA from tyrosine and DPHA from dopa by parsley Q06086. To
further verify the identity of the product, a tyrosine and parsley
Q06086 reaction mixture was derivatized with O-(4-nitroben-
zyl)hydroxylamine. The derivatized sample was injected onto an
RTX5MS GC column (30 m ꢀ 0.32 mm) installed in a HP 5890 GC.
2.2. Plant material, growth conditions, and jasmonic acid elicitation
Oven temperature was programmed from 80° to 280° at 8° minꢁ1
.
P. crispum seeds were obtained from www.burpee.com.
T. flavum seeds were obtained from www.dianeseeds.com. Seeds
were germinated in Sunshine Pro Premium potting soil and were
grown under a 16 h photoperiod at 23 °C at 100 microeinsteins.
Ten milliliters of a 2.5 mM Jasmonic acid (1.2% EtOH, 0.2% Triton
X-100) solution was injected into the soil surrounding the stem
of the parsley plant. In addition, 0.25 mL of 5 mM Jasmonic acid
solution was rubbed onto parsley leaves and stems.
EI mass spectrometry was performed on a VG70SE mass spectrom-
eter operated at 70 eV with the mass range scanned from 50–
400 amu. The molecular ion and its EI spectrum of the enzymatic
product derivative was compared with those of chemically synthe-
sized 4-HPAA-O-(4-nitrobenzyl) derivative obtained at identical
GC/MS analysis conditions. The 4-HPAA standard was chemically
synthesized from DL-4-hydroxyphenyllactic acid methanol. Sup-
plement material provides details about 4-HPAA synthesis/
derivatization.
2.3. RNA isolation, cDNA production, and RT-PCR
2.7. Kinetic analysis
Total RNA was isolated from whole parsley plants (12 weeks)
and T. flavum plants (12 weeks) using AmbionÒ mirVana™ miRNA
Isolation Kit. RNA samples were subsequently DNase-treated using
Ambion TURBO DNA-free™ Kit. cDNAs were produced using Invit-
rogen™ SuperScript™ III First-Strand Synthesis System for RT-PCR.
Specific parsley Q06086 forward (50-CTATGGAGTTGGTCAGCTGA
G-30) and reverse primer (50-CACCGACTCCAACAACTTC-30) and
control parsley polyubiquitin (ubi4) forward (50-CTTCGTCTCCGTG
GTGGT-30) and reverse primer (50-GCTAGGGTCCTTCCATCCTC-30)
were synthesized and used for RT-PCR with reagents and
procedures from Invitrogen™ PlatinumÒ SYBRÒ Green qPCR Super-
Mix-UDG. Each data point represented the average of three
independent biological samples with three technical replicates.
After the substrate specificity and catalytic reaction of parsley
Q06086 were verified, its kinetic parameter to tyrosine and dopa
were analyzed. Reaction mixtures of 100
parsley AAS (Q06086 protein) and
l
L containing 10
lg of
a
varying concentration
(0.1–5 mM) of tyrosine or dopa were prepared in 50 mM phos-
phate buffer (pH 7.0) and incubated at 25 °C. An equal volume of
borohydride-saturated ethanol was added to the reaction mixtures
at 5 min after incubation (stopped reaction by protein denatur-
ation and reduction of 4-HPAA to 4-HPEA by borohydride). The
mixtures were acidified by mixing equal volume of 0.8 M formic
acid (decomposition of remaining borohydride) and supernatants
were injected for HPLC-ED analysis. The amounts of products in
reaction mixtures containing different concentrations of substrate
were quantitated based on a standard curve generated using
authentic 4-PHEA standard (or DPHA standard) at identical condi-
tions of HPLC-ED analysis.
2.4. Protein expression and purification
A forward and reverse parsley Q06086 CDS primer pair (50-ACT
GACTAGTATGGGCTCCATCGATAATC-30 and 50-ACTGCTCGAGTTAGG
ATAAAATATTCACGATCTTCT-30) and a forward and reverse Thalic-
trum AAG60665 CDS primer pair (50-ACTGACTAGTATGGGTAGCC
TCCATGTT-30 and 50-ACTGCTCGAGTTAAAATGTAGCAAGTACAGCA
TC-30) were synthesized and used to amplify cDNA. Amplified
products were cloned into an Impact-CN protein expression vector
(New England Biolabs). All proteins were expressed and purified
according to the previous methods used in D. melanogaster DDC
recombinant production [3] with modifications. Protein concentra-
tion was determined using the Bradford method [4].
2.8. Analysis of endogenous tyrosine and dopa in parsley extracts by
LC/MSMS
Enzyme activity assays determined that parsley Q06086 could
use tyrosine and dopa as its substrates. To determine the presence
and relative amounts of tyrosine and dopa in parsley, whole adult
plants (12 weeks) were frozen and ground to homogeneity in li-
quid nitrogen using a mortar and pestle. Ground plant powder
was extracted with 80% ethanol at 1:25 (tissue powder weight ver-
sus volume of ethanol solution) for 30 min at room temperature.
The soluble fraction was then removed and lyophilized. The lyoph-
ilized samples were re-dissolved in 0.1% formic acid and analyzed
by a LC-3200 Q Trap MS/MS system (Applied Biosciences) at
positive ion mode. Identification of tyrosine and dopa in parsley
2.5. Activity assays
Reaction mixtures of 100
lL, containing 15 lg Q06086 or 5 lg
AAG60665 and 2 mM tyrosine or dopa, were prepared in 50 mM

M.P. Torrens-Spence et al. / Biochemical and Biophysical Research Communications 418 (2012) 211–216
213
extracts was based on their retention time and MS/MS spectra in
comparison with those of authentic tyrosine and dopa standards
at identical analytic conditions.
indicating that the broad peak corresponds to the reaction product.
Experimental repetition with dopa indicates a similar broad peak
corresponding to the dopa enzymatic product. When phenylala-
nine was used a substrate, the broad peak essentially was not ob-
served. This data was consistent with the previously reported
Q06086 substrate specificity [2]. Under identical HPLC-ED analysis
conditions, a sharp product peak was detected in a Thalictrum
AAG60665 protein and tyrosine (Fig. 2D) or dopa reaction mixture
(not shown).
3. Results
3.1. Spectral characteristics
Spectral analysis of purified recombinant parsley Q06086
protein and Thalictrum AAG60665 protein revealed the presence
of an ultraviolet (UV) absorbance peak with a kmax around
330–340 nm and a visible absorbance peak with a kmax around
398–422 nm (Fig. 1). These two absorbance peaks are typical for
PLP-containing proteins. The presence of the two absorbance peaks
indicated that parsley Q06086 and Thalictrum AAG60665 were
PLP-containing proteins. During ion exchange and gel filtration
chromatographies, no PLP was added to buffers used for protein
purification, indicating that PLP cofactor was tightly associated
with their polypeptides.
3.3. Product identification
The product peak in the recombinant Thalictrum AAG60665 pro-
tein and tyrosine reaction mixture was identified as tyramine
based on its coelution with tyramine standard at different chro-
matographic conditions. Production of dopamine in Thalictrum
AAG60665 protein and dopa reaction mixtures was verified by
the same procedures. Results demonstrate that Thalictrum
AAG60665 protein catalyzes the standard decarboxylation reaction
of tyrosine to tyramine and dopa to dopamine. This indicates that
AAG60665 gene encodes a true TyDC.
3.2. AAAD assays
The peak shapes resulting from parsley Q06086 protein incu-
bated with tyrosine or dopa were similar to the chromatographic
behavior of aromatic acetaldehyde produced by Drosophila DHPA
synthase [1]. This peak similarity suggests that the Q06086 en-
zyme might function in aromatic aldehyde synthase activity rather
than the previously reported aromatic decarboxylase activity.
Aldehydes can be reduced to their corresponding alcohol by boro-
hydride. When the incubated Q06086 and tyrosine reaction mix-
ture was treated with NaBH4 prior to HPLC-ED analysis, the
broad product peak (Fig. 2A and B) was converted to a sharp peak
(Fig. 2C) that coeluted with authentic 4-hydroxyphenylethanol (4-
HPEA) standard at different chromatographic conditions. When
incubated Q06086 protein and dopa was treated by NaBH4, the
broad product peak became a sharp peak that coeluted with 3,4-
dihydroxyphenylethanol (DHPEA) at different chromatographic
conditions (not shown).
When recombinant Q06086 protein was first assayed using
tyrosine as a substrate, a very broad peak was detected in the reac-
tion mixture by HPLC-ED (Fig. 2A). The peak dimension was pro-
portionally increased as the incubation time increased (Fig. 2B),
Parsley Q06086
422
339
The conversion of the broad product peak to a sharp peak by
borohydride (Fig. 2) and its coelution with authentic 4-HPEA pro-
vide sufficient basis to suggest the 4-HPAA identity of the broad
product peak (seen during HPLC-ED analysis of the parsley
Q06086 protein and tyrosine reaction mixtures). To further verify
the identity of the enzymatic product, incubated parsley Q06086
and tyrosine reaction mixtures were derivatized with O-(4-nitro-
benzyl)hydroxylamine and analyzed by GC/MS. Under the applied
GC/MS analysis conditions, a molecular ion of 286 was observed
and its EI spectrum displayed 77, 107, 133, 150 product ions
(Fig. 3C and D). The chemically synthesized 4-HPAA derivative pro-
duced the same molecular ion elution time and EI spectrum when
analyzed under identical conditions (Fig. 3A and B). The identical
molecular ion elution time and EI fragmentation pattern of the
derivatized tyrosine enzymatic product and the derivatized chemi-
cally synthesized 4-HPAA standard further proves Q06086s role in
the catalysis of tyrosine to 4-HPAA. These data clearly demonstrate
the true AAS identity of the previously reported parsley TyDC. Under
the applied conditions of kinetic analysis, parsley AAS showed a
340
360
400
420
440
380
nm
Thalictrum AAG60665
398
334
higher affinity to tyrosine (Km = 0.46 mM) than to dopa (Km
1.4 mM) and also higher Vmax to tyrosine (Vmax = 1.117
mol minꢁ1 mgꢁ1) than to dopa (Vmax = 0.318 mol minꢁ1 mgꢁ1).
=
a
l
l
320
400
340
420
360
380
3.4. Physiological substrate identification
nm
To determine the availability of physiological substrates within
parsley, LC/MS was utilized to measure the presence of internal
tyrosine and dopa levels. Assay development resulted in unique
multiple reaction monitoring (MRM) chromatographs for both
Fig. 1. Spectral characteristics of recombinant Q06086 and AAG60665 proteins.
Purified proteins were prepared in 50 mM phosphate buffer (pH 7.5) and their
absorbance spectrum were determined using a Hitachi U2001 UV Visible spectro-
photometer. Arrows indicate peak maximum.

214
M.P. Torrens-Spence et al. / Biochemical and Biophysical Research Communications 418 (2012) 211–216
A
B
C
D
Tyrosol
OH
HPAA
O
HPAA
O
Tyramine
NH2
OH
OH
OH
OH
Fig. 2. Q06086 and AAG60665 activity chromatograms. (A and B) illustrate the accumulation of 4-HPAA (the broad peak) in 0.1 mL reaction mixture containing 2 mM
tyrosine and 15 g Q06086 at 30 min (A) and 60 min (B) incubation. (C) shows the reduction of 4-HPAA to 4-HPEA by borohydride (the sample was prepared as in B, but it
was treated with NaBH4 before HPLC-ED analysis). (D) illustrates the production of tyramine in 0.1 mL reaction mixture containing 2 mM tyrosine and 5 g AAG60665 during
l
l
10 min incubation. Under identical conditions, the retention times of tyrosine, 4-HPEA and tyramine were 3.0, 5.1, and 6.9 min, respectively. The peak indicated by a red
arrow in B with a retention time of 8.2 min (that might be presumed as tyramine) was part of the broad peak and became a single 4-HPEA peak after borohydride reduction.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the tyrosine standard (Fig. 4A) and the dopa standard (Fig. 4C). The
assay was then utilized to measure tyrosine and dopa from whole
parsley extracts. Results demonstrated the presence of tyrosine
(Fig. 4B) and the absence of dopa (Fig. 4D). This suggests parsley
Q06086s potential role in 4-HPAA production and that tyrosine is
likely the sole physiological substrate for Q06086.
(PLP) dependence, and aromatic substrate requirements. Despite
their shared homology AAADs actually represent several distinct
enzyme groups. Each group is capable of catalyzing either the pro-
duction of monoamines or formation of aromatic acetaldehydes on
varied but stringent combinations of aromatic substrates. Variation
in product formation equates to diverse physiological functions.
This is particularly true for AAAD enzymes from plants and insects.
For example, some insect AAADs that use dopa as a substrate play
very different physiological roles as compared to those that use
tyrosine as a primary substrate [1,5,6]. Plant AAADs that catalyze
tyrosine to tyramine conversion play essential roles in the biosyn-
thesis of benzylisoquinoline alkaloids and many other secondary
metabolites [7–9] and those that catalyze the decarboxylation of
tryptophan and 5-HTP to tryptamine and 5-hydroxytryptamine
(5-HT), respectively, may regulate the biosynthesis of thousands
of indole alkaloid compounds [10–12]. Plant and insect AAADs that
catalyze the production of aromatic acetaldehydes have com-
pletely different functions as compared to those that produce aryl-
alkylamines [1,13,14]; as a result, their generic AAAD name
provides misleading functional implications.
3.5. Potential function of parsley AAS in defense/stress responses
Q06086 was previously characterized as a pathogen-responsive
gene because fungal infection or elicitor treatment led to a rapid
increase in its gene transcription in parsley cell suspension culture
[2]. To verify if Q06086 is a defense/stress responsive gene in
whole plants, parsley plants were treated with jasmonic acid (JA)
for a 10, 24, and 48 h time period. RT-PCR analyses of Q06086 tran-
script levels in comparison to polyubiquitin (ubi4) demonstrate a
fourfold transcript increase after 10 h, a 2.9-fold transcript increase
at 24 h, and a return to control transcript level at 48 h. Up-regula-
tion of the Q06086 transcription by JA suggests that it is a stress/
defense responsive gene.
To link AAADs with functionally relevant information, efforts
have been made to further classify plant and insect AAADs accord-
ing to their substrate specificity. Due to high level of sequence
homology of AAADs across species, annotation through primary se-
quence comparison is highly inaccurate. For example, until re-
cently insects AAS proteins have been considered as DDC
isozymes [1]. Comparison of Q06086 and AAG60665 especially
exemplifies the sequence ambiguity of AAADs in plants. Despite
different catalytic reactions and broad evolutionary divergence
Q06086 and AAG60665 retain 70% sequence identity and 83% se-
quence homology. Evaluations of Q06086 and AAG60665 respec-
tive spectra further illustrate their conformity. In AAADs, the PLP
cofactor is associated with their proteins by a Schiff-base linkage
3.6. Parsley extract 4-HPAA measurements
GCMS analysis was used to measure internal levels of 4-HPAA
from non-elicited plants and JA elicited plants. The GCMS assay
developed to measure the chemically synthesized 4-HPAA deriva-
tive was utilized to measure derivatized 4-HPAA in extracts from
both non-elicited plants and JA elicited plants. When parsley ex-
tracts were analyzed, no 4-HPAA was detected in either the non-
elicited sample or the JA elicited sample. This absence of 4-HPAA
may indicate that the 4-HPAA formed in parsley is rapidly con-
verted to downstream compounds.
formed between the aldehyde group of PLP and the e-amino group
4. Discussion
of a conserved active site lysine. Because this linkage is universal
for all PLP-containing decarboxylases, the differences in spectral
characteristics among distinct AAADs suggest some differences in
the active site residues surrounding the PLP cofactor. Since
In protein databases, proteins of the AAAD family are grouped
together through their high homology, pyridoxal-50-phosphate

M.P. Torrens-Spence et al. / Biochemical and Biophysical Research Communications 418 (2012) 211–216
215
A
B
B
A
23:50
107
3:36
100%
100%
2.0e4
10000
3:36
75
50
25
0
75
50
25
0
133
1.0e4
0.0
5000
0.0
77
78
150
151
106
286
C
D
2.0e4
1.0e4
0.0
10000
3:00
3:00
300
60
120
180
240
C
D
23:51
107
5000
100%
100%
75
50
25
75
50
25
0
133
0.0
77
78
Fig. 4. Parsley extract LCMS chromatograms of tyrosine and dopa. (A and C)
represent the respective chromatography peaks resulting from select MRM ions of
tyrosine and dopa standards. (B and D) represent the respective chromatography
peaks resulting from select MRM ions of tyrosine and dopa measured from parsley
metabolite extracts. This figure illustrates the relative abundance of tyrosine and
absence of dopa with in parsley metabolite extracts. The y axis represents intensity
in cps.
150
151
106
286
0
We previously proposed that the chromatographic behavior of
the DHPA peak during HPLC-ED analysis was due to the keto-enol
tautomerization [1]. The same phenomenon might also be responsi-
ble for the odd peak dimensions observed for 4-HPAA in this study.
The chromatographic behavior of benzaldehyde, 4-hydroxybenzal-
dehyde and 3,4-dihydroxybenzaldehyde supports this suggestion.
These aromatic aldehydes elute as sharp peak during HPLC-ED
analysis under similar conditions and treatment of these com-
pounds with NaBH4 reduced them to their corresponding aromatic
alcohols. In these aromatic aldehydes, formation of their side chain
enol tautomers requires a series electron shift of their benzene,
phenyl or catechol ring, which may not be favored. In contrast, the
keto-enol tautomerization does not cause apparent change of the
ring structure in DHPA and 4-HPAA, which may proceed relatively
easily under acidic conditions during reverse-phase separation.
The instability/reactivity of the 4-HPAA product might be partly
responsible for the misidentification of Q06086. This also might ex-
plain in part why plant and insect AASs were identified just re-
cently [1,13] (while other types of AAADs have been established
for many years [16,17]). In a previous study, we illustrated the
instability of DHPA produced by insect AAS with dopa as a sub-
strate [1]. The commercial availability of the aromatic aldehydes
and the absence of the commercial aromatic acetaldehydes further
imply the reactivity/instability of the aromatic acetaldehydes.
There have been many reports that discussed the toxicity of DHPA
in the development of human neurodegenerative diseases [18,19].
The chemical would have been available as a standard (because
there is a need for clinical studies) if it were stable after synthesis.
The odd peak dimensions might also contribute to AAS misidenti-
fication. Depending upon the amount of enzyme incorporated into
the reaction mixture and the incubation time, the shallow broad
peak we defined in this work could be easily presumed to be base-
line noises due to contaminants from substrate or protein samples.
300
60
120
180
240
Fig. 3. GC–MS chromatograms and ions spectra for chemically and enzymatically
synthesized 4-HPAA derivatives. (A) represents the total ion count chromatography
peak resulting from the chemically synthesized derivatized 4-HPAA. (B) represents
the spectra resulting from the chemically synthesized derivatized 4-HPAA. (C)
represents the total ion count chromatography peak resulting from the enzymat-
ically synthesized derivatized 4-HPAA. (D) represents the spectra resulting from the
enzymatically synthesized derivatized 4-HPAA. The ion spectra from both products
B and C are highly similar. (B and C) illustrate key ions including the molecular ion
(286) and strong product ions (77, 107, 133, 150). Ions below the 25% mark were
not included to maintain figure clarity.
comparison of Q06086 and AAG60665 (Fig. 1) yield minor varia-
tions, one must speculate that their respective PLP surrounding
residues are highly similar. This spectral similarity illustrates that
the Q06086 vs AAG60665 activity differentiating resides are truly
quite subtle.
Another major problem in plant AAAD classification also in-
cludes the presence of incorrectly identified distinct plant AAAD
proteins. Our initial intention in this study was to utilize Q06086
as a TyDC standard (because it has been experimentally verified
[2]) to compare with AAS enzymes. Characterization work with
Q06086 ultimately led us to its identification as a plant AAS. An
extensive literature search suggests that misidentification of plant
AAAD might be due to the method used for AAAD activity assays.
Detection of CO2 release has commonly been used in AAAD assays
[2]. The method is valid once the decarboxylative nature of the
reaction is established, but CO2 release itself cannot distinguish
AAS activity from TyDC activity. This may have been partly respon-
sible for some plant AAAD misidentifications. For example, the first
reported Cytisus scoparius DDC about 40 years ago [15] likely is an
AAS because oxygen is necessary for CO2 release by the enzyme
(oxygen is required for oxidative deamination by AASs).

216
M.P. Torrens-Spence et al. / Biochemical and Biophysical Research Communications 418 (2012) 211–216
[2] P. Kawalleck, H. Keller, K. Hahlbrok, et al., A pathogen-responsive gene of
Q06086 was previously proposed to be a defense response gene
parsley encodes tyrosine decarboxylase, J. Biol. Chem. 268 (3) (1993) 2189–
2194.
[3] Q. Han, H. Ding, H. Robinson, et al., Crystal structure and substrate specificity
because fungal infection or fungal extract treatment up-regulated
its transcription in about an hour [2]. We did observe a similar sig-
nificant up-regulation Q06086 transcription when JA was applied
to the plant. This provides a basis to suggest that this AAS is a de-
fense/stress responsive gene because JA regulates plant responses
to abiotic and biotic stresses [20]. An apparent increase in AAS
transcription should lead to an increased level of the AAS protein,
which should in general be reflected by the increased content of 4-
HPAA in parsley. Despite this rational, analysis of parsley extracts
from both elicited or control samples did not result in the detection
of 4-HPAA. Data from this study allows us to propose a positive
relationship between Q06086 expression and defense/stress condi-
tions of the parsley plant. The overall specific functions of this
intriguing enzyme remain to be substantiated.
In summary, results from this study provide solid evidence
demonstrating that the previously identified Q06086 parsley TyDC
is a plant AAS that uses tyrosine as a primary substrate. Up-regula-
tion of Q06086 transcription by JA indicates that Q06086 is a de-
fense/stress responsive gene. Although details concerning the
molecular regulation of the Q06086 gene and the precise functions
of its protein remain to be established, our data provide a founda-
tion towards achieving a comprehensive understanding of the bio-
chemistry and molecular biology of this interesting protein. In
addition, the aforementioned reactivity and instability of aromatic
acetaldehydes should serve as a useful reference in studies dealing
with the activity of similar AAS proteins.
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Acknowledgment
This study was supported through Virginia Tech Biochemistry
college of Agricultural and Life Sciences funding.
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