An unnatural amino acid based fluorescent probe for phenylalanine ammonia lyase

Article abstract of DOI:10.1039/c4ob00914b

A fluorescent probe (2a-LP) based on an unnatural amino acid (UAA) is developed for the detection of phenylalanine ammonia lyase (PAL). In the presence of PAL, 2a-LP is catalytically deaminated to ortho-amino-transcinnamic acid (o-a-CA), which shows a rem

Full text of DOI:10.1039/c4ob00914b

Organic &
Biomolecular Chemistry
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An unnatural amino acid based fluorescent probe
for phenylalanine ammonia lyase†
Cite this: Org. Biomol. Chem., 2014,
12, 5818
Zhenlin Tian, Weiping Zhu,* Yufang Xu* and Xuhong Qian
Received 4th May 2014,
Accepted 17th June 2014
DOI: 10.1039/c4ob00914b
www.rsc.org/obc
A fluorescent probe (2a-LP) based on an unnatural amino acid activity.6a For example, β-glucuronidase (GUS) or luciferase repor-
(UAA) is developed for the detection of phenylalanine ammonia ter enzymes have been used to examine PAL gene expression in
lyase (PAL). In the presence of PAL, 2a-LP is catalytically deami- transgenic tobacco, bean and A. thaliana.6b Rookes reported a
nated to ortho-amino-transcinnamic acid (o-a-CA), which shows a GFP probe for PAL.6c Compared to conventional HPLC assays
remarkable “off–on” fluorescence signal. Thus, the probe 2a-LP and GFP probes, small-molecular fluorescent probes are widely
enables direct visualization of the PAL activity in tomato under UV used in detecting, tracing, and visualizing the function of
illumination and has potential in vitro assays.
enzymes because of their insignificant steric bulk, fast labelling
kinetics and characteristics such as real-time determination,
high selectivity and sensitivity.7 Thus, developing specific fluo-
rescent probes for PAL is of considerable interest.
Deamination of L-Phe by PAL results in efficient conju-
gation between the carboxyl and the benzene ring. We envi-
sage that incorporation of an electron donating group at the
ortho/para position of the benzene ring will build an electronic
push–pull system, which is typically a good dye and hopefully
a good fluorophore. Based on the aforementioned design
rationale, we synthesized an L-Phe derivative, 2-amino-
L-phenylalanine (2a-LP), as a PAL probe (Scheme 1).
As shown in Scheme 1, upon deamination by PAL, 2a-LP
would release an ortho-amino-transcinnamic acid (o-a-CA),
which emits blue fluorescence upon UV excitation. Utilizing
this unnatural amino acid (UAA) as a PAL substrate, the PAL
activity can be monitored. This is the first report using a syn-
thetic UAA as a fluorescent probe for the direct detection of
PAL activity. Previously, UAAs with unique small bioorthogonal
handles have been frequently used in site-specific labelling of
biomacromolecules,8 and this new UAA based enzyme probe
broadens the scope of applications of UAAs.
Phenylalanine ammonia-lyase (PAL) is
a non-hydrolytic
enzyme and catalyses the biotransformation of L-phenylalanine
(L-Phe) to trans-cinnamic acid (t-CA) and ammonia in the
absence of exogenous cofactors.1a PAL is widely distributed in
higher plants, fungi, yeasts and prokaryotes (Streptomyces), but
absent in eukaryotic bacteria and animal tissues.1b PAL is
employed in enzyme replacement therapy (ERT) for phenyl-
ketonuria (PKU).2 PAL plays an important role in the catabo-
lism of microorganisms, allowing them to use L-Phe as a sole
source of carbon and nitrogen.3 PAL is also involved in the bio-
synthesis of salic acid (SA) and phenylpropanoids, which play
fundamental roles in plant immune response, development
and protection against environmental stresses.4a,b Expression
of PAL is rapidly induced during plant–pathogen interactions,
and inhibition of PAL activity results in its breakdown.4c The
determination of PAL activity is therefore of significance and
can be used to monitor the plant induced resistance (IR).
PAL activity is typically estimated by the level of t-CA, which
is measured by UV-Vis spectroscopy. However, coumarin based
compounds have similar absorption maximum to t-CA and con-
stitute strong basal interferences.5 High performance liquid
chromatography (HPLC) is a robust alternative for t-CA measure-
ment. Another approach is to examine PAL gene expression. One
usually uses specific reporter molecules, which are induced
upon the activation of the PAL promoter, to analyse the PAL
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of
Chemical Biology, School of Pharmacy, East China University of Science and
Technology, 130 Meilong Road, Shanghai, 200237, PR China.
E-mail: wpzhu@ecust.edu.cn, yfxu@ecust.edu.cn; Fax: +86 21-64252603
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob00914b
Scheme 1 Proposed mechanism of 2a-LP as PAL probe.
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2a-LP and o-a-CA were synthesized as shown in Schemes
S1–S2 (ESI†), and their structures were confirmed from 1H
NMR, 13C NMR, and HRMS spectra (Sections S2 and S3, ESI†).
The absorption and fluorescence spectra of the probe and
its enzymatic products were measured. o-a-CA emitted fluo-
rescence at 494 nm with excitation at 335 nm and showed a
159 nm Stokes shift, and had similar fluorescence properties
to trans-2-amino-5-hydroxymethyl-cinnamic acid.9 The pH
titration of o-a-CA showed that its fluorescence is stable
between pH values of 6 and 10. The fluorescence quantum
yield and the pKa of o-a-CA were calculated to be 0.106 and 4.7
in water, respectively (Table S1, Fig. S2, ESI†). Since 2a-LP is
non-fluorescent, a significant “off–on” signal is expected if
2a-LP can be catalysed and deaminated by PAL.
Then the probe, 2a-LP was tested to detect the PAL activity
in buffer solution (Tris-HCl, 50 mM). As the commercial PAL
(Rhodotorula glutinis source) works at an optimal pH of 8.5,
0.5 mM Tris-HCl (pH 8.5) buffer solution was used in the sub-
sequent experiments. 2a-LP was incubated with 0.132 U mL−1
PAL solution for 10 h at 30 °C and the fluorescence intensity
was measured. The fluorescence intensity of the above solution
showed 13-fold enhancement after 2 h, and can be visualized
by the naked-eyes under illumination with an UV lamp. In
order to estimate the conversion of 2a-LP, we run the assay
and plot fluorescence intensity versus concentration of o-a-CA
for each concentration (Fig. S3, ESI†). It was showed that
35.2% of 2a-LP had been deaminated after 2 h, and 98.0% of
2a-LP had been consumed after 10 h. The results demon-
strated that 2a-LP could be converted by PAL efficiently (Fig. 1).
Furthermore, the enzymatic metabolite of probe, 2a-LP was
analyzed by HPLC (Fig. 2). As expected, the retention peak of
probe’s enzymatic product is consistent with that of o-a-CA, and
the retention time of o-a-CA and enzymatic product of 2a-LP is
10.08 min and 10.05 min, respectively. The results further con-
firmed the proposed mechanism of 2a-LP as a PAL probe.
Fig. 2 Reversed-phase HPLC analysis of the PAL catalyzed deamination
reaction of 2a-LP. Conditions: 50 mM Tris-HCl buffer (pH 8.5), detected
by UV 235 nm. (Red line) 0.5 mM 2a-LP in Tris-HCl buffer; (blue line)
0.5 mM 2a-LP and 0.5 mM o-a-CA in Tris-HCl buffer; (black line) 1 mM
2a-LP incubated with PAL in Tris-HCl buffer for 5 h.
Next, we investigated whether the progress of the enzymatic
reaction of 2a-LP could be kinetically monitored by fluorome-
try. The fluorescence spectra of the 2a-LP were periodically
recorded during incubation of 2a-LP with PAL, and the con-
sumption of 2a-LP by PAL led to a progressive increase in fluo-
rescence intensity which could be followed in a continuous
manner (Fig. S4, ESI†). Behaviours of 2a-LP towards PAL show
that fluorescence intensity is proportional to the enzyme
activity within two hours’ incubation (Fig. S5, ESI†). To esta-
blish a method for rapid detection of PAL activity, fluorescence
intensity after one hour’s incubation was chosen to establish a
calibration curve (Fig. 3a). Combining the calibration curve
and the fluorescence spectrum of an unknown sample incu-
bated with 2a-LP, the PAL activity could be estimated quickly.
With PAL, the enzymological properties of 2a-LP have been
studied using a continuous assay. After confirming the linear-
ity of the integrated fluorescence response caused by various
concentrations of o-a-CA (Fig. S3, ESI†), it was found that the
initial reaction velocities were proportional to the PAL concen-
tration (Fig. S6, ESI†). Then a suitable enzyme/substrate ratio
was established, and the substrate concentration was varied to
generate a saturation curve for determining the Km value of
2a-LP. The alternation of the initial velocity with the concen-
tration of 2a-LP was demonstrated by the Michaelis–Menten
equation and the Lineweaver–Burk plot, and the result was cal-
culated as Km of 1.570 mM and Vmax of 0.245 μM min−1
(Fig. 3b). This Km value of 2a-LP is 2.4-fold of its natural sub-
strate, and the para-substituted L-Phe has smaller Km values
(Table S2, ESI†). This might be due to the fact that ortho-amino
in 2a-LP interfered with binding with a 4-methylidene-imidazole-
5-one group (MIO) cofactor of PAL, and the better affinities of
para-substitutes also provide us with further study orientation.
Next, 2a-LP was used to monitor the activity of PAL in plant
samples. Ultraviolet A (UV-A) induced plant samples and blank
Fig. 1 Time course of fluorescence spectra of probe in the presence of
PAL. Conditions: 2a-LP (5 μM) with PAL (0.132 U mL) in 50 mM Tris-HCl
buffer (pH 8.5), λex = 335 nm, slit: 10/10 nm. Spectra were measured
automatically every 10 min.
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Fig. 4 Fluorescence responses of probe with tomato samples. Con-
ditions: incubated 2a-LP (5 μM) with samples for 1 h at 30 °C, λex
=
335 nm, λem = 494 nm, slit = 10/10 nm. Black column: the PAL extraction
was from non-treated tomatoes without addition of probe, red column:
incubated 2a-LP with the PAL extraction from none UV treated toma-
toes, blue column: incubated 2a-LP with the PAL extraction from UV
treated tomatoes, dark green column: incubated 2a-LP with the PAL
extraction from UV treated tomato with addition of L-Phe (5 μM).
enhancement in the UV irradiated sample was caused by the
catalytic activity of PAL. This result demonstrates that 2a-LP has
great potential in the detection of PAL activities in plant samples,
which is of significance in plant induced resistance research.
PAL has also been an important ERT tool for PKU,2c moni-
toring PAL activity under physiological conditions is of great
significance for PKU ERT research. Therefore, we also explored
the potential applications of 2a-LP in biological systems. Incu-
bation of HeLa cells with PAL for 3 hours at 37 °C was followed
by the addition of 2a-LP or none, and then incubation for
another 30 min. As shown in Fig. 5, HeLa cells with only PAL
Fig. 3 (a) Fluorescence calibration curve for PAL activity. Probe (5 μM),
PAL (0.026 U mL−1, 0.050 U mL−1, 0.071 U mL−1, 0.092 U mL−1, 0.132 U
mL−1), the estimated linear equation is y = 207.6x + 8.5 (r2= 0.994). (b)
Fluorimetric assay of PAL and 2a-LP: Michaelis–Menten and Line-
weaver–Burk plot (insert). PAL (0.012 U mL−1), 2a-LP (20–10 000 μM).
Initial velocity values were calculated from the reaction time courses
and based on the respective fluorescence signal. Initial velocity values
were fitted to the Michaelis–Menten plot and to the Lineweaver–Burk
plot, estimated linear equation y = 6402.638x + 4.0778, r2 = 0.979).
Other conditions: Tris-HCl buffer (50 mM, pH 8.5), 30 °C, λex = 335 nm,
λem = 494 nm, slit: 10/10 nm.
plant samples were designed because ultraviolet UV-A is an
environmental stimulus, which can increase the expression of
PAL gene in tomato.10 2a-LP was applied in detection of the
PAL activity in the UV-A irradiated tomato samples (Section S2,
ESI†). 2a-LP (5 μM) was incubated with UV-A irradiated and
non-irradiated samples, and it was observed that the fluo-
rescence intensity of UV-A irradiated samples showed 3.3-fold
enhancement than that of non-treated samples (Fig. S7, ESI†).
Combining the increased intensity after 1 hour with the cali-
bration curve, we estimated the PAL activity of the UV-A ir-
radiated sample as 0.035 U mL−1, and the non-UV treated
sample showed no difference with the negative control (Fig. 4).
Also the UV-A irradiated sample mixed with L-Phe (5 μM) has
been tested and it showed no obvious fluorescence intensity
change (Fig. 4). This result might be due to the interference of
L-Phe. L-Phe acts as a competitive inhibitor hindered 2a-LP
binding with PAL, it also confirmed that the fluorescence
Fig. 5 Image of PAL detection in HeLa cells using 2a-LP at 37 °C. (a)
Bright-field image of HeLa cells incubated with PAL for 3 h. (b) Bright-
field image of HeLa cells incubated with PAL for 3 h with 2a-LP
(100 mM) added for the final 30 min. (c) Fluorescence image of HeLa
cells incubated with PAL for 3 h. (d) Fluorescence image of HeLa cells
incubated with PAL for 3 h with 2a-LP (100 mM) added for the final
30 min.
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showed low fluorescence, while in the presence of PAL and 2a-
LP, HeLa cells show strong fluorescence, which also suggest
that PAL can be monitored intracellularly by 2a-LP. This result
demonstrates that 2a-LP has potential in visualizing PAL level
changes of living cells, and 2a-LP can be a novel tool for PKU
ERT research.
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Conclusions
In summary, we have developed a new fluorescent probe, 2a-
LP, based on the modification of PAL natural substrate, L-Phe,
for PAL activity. Enzymatic deamination can be monitored
upon mixing the probe and samples, without any additional
treatment. Successful monitoring of this enzymatic activity is
realized by utilizing the UAA deamination mechanism for the
first time. This probe can be applied to monitor PAL activity in
both plant samples and HeLa cells, and perhaps be a promis-
ing tool for the detection of PAL activity and plant induced
resistance research.
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Acknowledgements
We thank the financial support from the National Science and
Technology Pillar Program of China (2011BAE06B02), the
National Natural Science Foundation of China (21236002), the
National Basic Research Program of China (973 program,
2013CB733700, 2010CB126100), the National High Technology
Research and Development Program of China (863 program,
2011AA10A207), the Shanghai Pujiang Program and the Funda-
mental Research Funds for the Central Universities. We also
thank Dr Charles Yang for the improvement of this article.
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