A simple bioluminescent method for measuring d-amino acid oxidase activity

Article abstract of DOI:10.1039/c4cc08145e

d-Amino acid oxidase (DAO) plays important roles in regulating d-amino acid neurotransmitters and was recently identified as a key enzyme integral to hydrogen sulfide production from d-Cys. We report here the development of a simple biocompatible, bioluminescent method for measuring DAO activity based on the highly selective condensation of d-Cys with 6-hydroxy-2-cyanobenzothiazole (CBT-OH) to form d-luciferin. This journal is

Full text of DOI:10.1039/c4cc08145e

ChemComm
View Article Online
View Journal
COMMUNICATION
A simple bioluminescent method for measuring
D-amino acid oxidase activity†
Cite this:DOI: 10.1039/c4cc08145e
T. Spencer Bailey,‡ Micah T. Donor,‡ Sean P. Naughton and Michael D. Pluth*
Received 14th October 2014,
Accepted 30th October 2014
DOI: 10.1039/c4cc08145e
www.rsc.org/chemcomm
D-Amino acid oxidase (DAO) plays important roles in regulating D-amino acid oxidase (DAO), which oxidatively deaminates
1
1
D-amino acid neurotransmitters and was recently identified as a key D-amino acids to their corresponding a-keto acids. Typically
enzyme integral to hydrogen sulfide production from D-Cys. We report involved in neurochemical regulation of D-amino acid neuro-
here the development of a simple biocompatible, bioluminescent transmitters in the brain, DAO metabolizes D-Cys to form
method for measuring DAO activity based on the highly selective 3-mercaptopyruvate (3-MP), which is a substrate for 3-MST
2
condensation of D-Cys with 6-hydroxy-2-cyanobenzothiazole (Scheme 1), thus connecting DAO with H S synthesis. Impor-
(
CBT-OH) to form D-luciferin.
tantly, administration of D-Cys has been shown to elevate sulfane
sulfur levels more effectively than L-Cys, suggesting a possible
1
0
Sulfur-containing compounds constitute a diverse palette of therapeutic potential for D-Cys derived H S. Despite the impor-
2
biologically-important molecules that play central roles in tance of DAO in neurochemical regulation and H S production,
2
signaling and homeostasis. Among these species, hydrogen measuring DAO activity under non-invasive conditions remains
1
2
2
sulfide (H S) has emerged as an important biological transmitter a significant challenge. For example, downstream detection of
contributing to signaling processes supporting physiological 3-MP or other a-oxy acids is not a specific assay for DAO activity
functions ranging from long term potentiation in the central because these a-oxy acids are also produced by other enzymes.
1
nervous system to vasorelaxation in the cardiovascular system.
To address this unmet need, we report here a simple bio-
À
Most prevalent as HS at physiological pH, sulfide can also be luminescent method based on firefly luciferase for measuring
stored in acid-labile, and reductant-labile pools. Emerging D-Cys levels and DAO activity.
2
evidence suggests that reductant-labile sulfane sulfur, which
Bioluminescence is a well-studied reporting method readily
includes persulfides (RSSH) and polysulfides (RS(S) SR), is an used for bioimaging with small molecule probes and quantita-
n
1
3,14
important post-translation modification of Cys residues involved tive measurements in ELISA assays.
in altered protein function, cellular signalling, and sulfide common substrate used in bioluminescence studies, we chose
Because D-luciferin is a
3
,4
5
–9
storage.
2
Most biosynthetic H S is produced enzymatically to use D-Cys as a substrate in our investigations because it can
from Cys and Hcy by the pyridoxal-5-phosphate (PLP) dependent be metabolized by DAO but can also react with 6-hydroxy-
enzymes cystathionine-b-synthase (CBS) and cystathionine-g- 2-cyanobenzothiazole (CBT-OH) to form D-luciferin. Similar con-
lyase (CSE) as well as L-cysteine aminotransferase (CAT) working densations of 1,2-aminothiols with 2-cyanobenzothioazoles have
together with the PLP-independent enzyme 3-mercaptopyruvate been previously applied as a template for polymer aggregation to
1
15
sulfurtransferase (3-MST). Recent reports now suggests that a monitor free Cys and homocysteine, to monitor caspase activity
16
fourth enzymatic pathway, primarily active in the cerebellum in the presence of peroxide, and to develop protein labeling
1
0
and kidneys, generates H
supported by the observation that treatment of neuronal tissues proteins
with D-Cys results in faster and more efficient H S production in live cell assays. Based on this reactivity and because D-luciferin
2
S from D-Cys. These findings are strategies for genetically encoded 1,2-aminothiol residues in
17,18
allowing for fluorescent and colorimetric imaging
2
1
0
than treatment with L-Cys.
H
2
S production from D-Cys relies on generates a bioluminescent signal when metabolized by firefly
luciferase, we envisioned that treatment of DAO with a known
Department of Chemistry and Biochemistry, Institute of Molecular Biology,
Materials Science Institute, University of Oregon, Eugene, OR, 97403, USA.
E-mail: pluth@uoregon.edu
concentration of D-Cys, followed by quenching of unreacted D-Cys
with CBT-OH to generate D-luciferin, would provide a simple
method for measuring DAO activity.
For this strategy to be biologically compatible, the reaction
of CBT-OH with internal Cys residues in proteins or peptides
†
Electronic supplementary information (ESI) available: Experimental methods,
UV-vis data. See DOI: 10.1039/c4cc08145e
These authors contributed equally to this work.
‡
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun.

View Article Online
Communication
ChemComm
Scheme 1 Routes for H
2
S synthesis from 3-MST and the associated strategy for using D-Cys to measure DAO activity using bioluminescence. DAO:
D-amino acid oxidase; CAT: L-cysteine aminotransferase; 3-MST: 3-mercaptopyruvate sulfurtransferase; P. Pyr: Photinus pyralis (firefly luciferase).
must be reversible to ensure that CBT-OH is not scavenged by is used in place of NAC the conversion of CBT-OH to luciferin is
GSH or other cellular nucleophiles. Additionally, CBT-OH must slower, however, the luciferin product remains the primary
react quickly and irreversibly with free D-Cys to generate the reaction product (Fig. S3, ESI†). These results establish that
D-luciferin product. To establish this feasibility, we investigated internal thiols in peptides or proteins should not interfere in
1
the reversible addition of thiols to CBT-OH by H NMR spectro- the detection of Cys by CBT-OH.
scopy using N-acetyl cysteine (NAC) as a substrate. Because NAC
Having demonstrated that CBT-OH forms luciferin in the
lacks a free amine group, nucleophilic addition to the electro- presence of multiple thiols, we next investigated whether
philic CBT-OH generates an imidothioate that cannot cyclize to the condensation rate of CBT-OH with Cys is fast enough to
form the luciferin product. Treatment of CBT-OH with NAC be a viable bioassay method. To measure the rate of reaction of
resulted in complete consumption of the CBT-OH starting CBT-OH with Cys, we treated CBT-OH with different concentra-
material and generation of new aromatic peaks in the tions of excess Cys under pseudo first-order conditions and
1
H NMR spectrum, indicating complete reaction of CBT-OH monitored the reaction by UV-vis spectroscopy (Fig. S1, ESI†).
with NAC (Fig. 1b and c). Addition of one equivalent of Cys to Based on these kinetic experiments, we determined that the
the CBT-OH–NAC reaction mixture immediately generates new reaction is first-order in Cys and determined an overall second-
À1 À1
aromatic peaks as well as a characteristic peak at 5.4 ppm order rate constant of 14.9 M
s
for the reaction (Fig. S2,
corresponding to luciferin (Fig. 1d). Equilibration of the reac- ESI†), which is similar to the previously reported value of
À1 À1
tion mixture results in complete conversion of the NAC-adduct 9.19 M
s
for 1,2-aminothiol condensations with cyano-
1
7
to the luciferin product, confirming the reversibility of NAC benzothiazoles. This condensation is almost 200 times faster
addition (Fig. 1e). Further treatment of the luciferin product than the commonly-used azide/cycloalkyne click reaction
1
À2
À1 À1 19
with NAC failed to produce changes in the H NMR spectrum, (7.6 Â 10
M
s ), suggesting that the CBT-OH/Cys con-
thus confirming the irreversibility of Cys addition. When GSH densation reaction is kinetically viable for bioconjugation.
Having investigated the reaction conditions under pseudo
first order conditions, we further explored the suitability of
the system for measuring D-Cys concentrations by treating
100 mM CBT-OH with sub-stoichiometric amounts of D-Cys
and analyzing the resulting bioluminescent signal. Condensa-
tion of CBT-OH with D-cysteine enables us to detect changes in
bioluminescent signal at resulting luciferin concentrations as
low as 293 nM. After incubation for 1 hour to allow for
conversion of CBT-OH to D-luciferin, introduction of luciferase
resulted in a bioluminescent response that linearly correlated
with D-Cys concentration (Fig. 2b).
Based on the linear bioluminescence response with D-Cys
concentration, we next used the CBT-OH system to measure
DAO activity. Because DAO metabolizes D-Cys to 3-MP, intro-
duction of an excess of CBT-OH at different time points in the
reaction of DAO with D-Cys should convert any remaining,
unreacted D-Cys to D-luciferin, which can then be measured
by addition of luciferase. To test this design, 20 mM of D-Cys was
Fig. 1 Selectivity of CBT-OH for Cys. (a) CBT-OH reacts reversibly
with non-Cys thiols, but irreversibly with Cys. H NMR spectra (500 MHz,
À1
incubated with 0.1 mg mL
DAO, and the reaction was
1
quenched at different time points with an excess of CBT-OH.
Addition of luciferase and measurement of the resultant bio-
luminescence from each sample revealed a rapid decrease in
d
6
-DMSO–D
2 3
O–CD OD mixture, room temperature) of (b) CBT-OH;
(c) after equilibration with 1 equiv. of N-acetyl cysteine (NAC); (d) immediately
after addition of Cys; and (e) after equilibration.
Chem. Commun.
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
the observed bioluminescence signal, correlating with a
decrease in D-Cys concentration upon metabolism by DAO
(Fig. 3a, black squares). By contrast, use of L- instead of D-Cys
did not generate a bioluminescence response, confirming that
the L-luciferin condensation product is not a bioluminescent
substrate for luciferase. Similarly, if D-Cys is incubated with
CBT-OH in the absence of DAO, the bioluminescence stays
constant at each time point, confirming a constant concen-
tration of D-Cys in the absence of DAO (Fig. 3a, blue triangles).
To further demonstrate that the developed method was report-
ing on DAO activity, we treated DAO with sodium benzoate, a
À6
11
competitive inhibitor of DAO, (K = 2.0 Â 10 M) in the
i
presence of D-Cys and measured the bioluminescent response
(Fig. 3b). As expected, a significantly higher concentration of
D-Cys remained during the assay, confirming that DAO activity
was reduced. We also tested the developed assay to probe the
affinity of other D-amino acids for DAO. For example, treatment
of DAO with equimolar amounts of D-Cys and D-serine did not
change the rate of D-Cys metabolism, suggesting that D-Cys is a
Fig. 2 (a) Detection of D-Cys by condensation with CBT-OH followed
by treatment with P. pyralis generates a bioluminescence response;
(
b) Bioluminescent response measured with varying concentrations of
D-Cys incubated with 100 mM CBT-OH for 1 h followed by addition of
À1
0
.1 mg mL P. pyralis. The bioluminescence was integrated at 560 nm for better substrate for DAO than D-serine.
2+
4
5 min in 10 mM Mg , 2 mM ATP, pH 7.4 tris buffer (50 mM), 37 1C.
In conclusion, the condensation reaction of CBT-OH with
Cys provides simple bioluminescent method for measuring
D-Cys. We also demonstrated that this condensation reaction
can also be used to monitor DAO activity by measuring
unreacted D-Cys as a function of time and/or potential DAO
inhibitors. Based on the availability of cell lines and animal
models that express luciferase enzymes, we envision that this
bioluminescent approach for investigating DAO activity and
function will provide a useful platform to inform on emerging
2
the roles of DAO in neurochemistry and H S signaling.
We thank Prof. Brad Nolen and his lab for generous use of
their plate reader. This work was supported by the NIGMS
(R00GM092970) and the University of Oregon (UO). The NMR
facilities at the UO are supported by NSF/ARRA (CHE-0923589).
Notes and references
1
2
R. Wang, Physiol. Rev., 2012, 92, 791–896.
X. Shen, E. A. Peter, S. Bir, R. Wang and C. G. Kevil, Free Radical Biol.
Med., 2012, 52, 2276–2283.
3
4
5
Y. Kimura, Y. Mikami, K. Osumi, M. Tsugane, J.-i. Oka and
H. Kimura, FASEB J., 2013, 27, 2451–2457.
S. Koike, Y. Ogasawara, N. Shibuya, H. Kimura and K. Ishii, FEBS
Lett., 2013, 587, 3548–3555.
A. K. Mustafa, M. M. Gadalla, N. Sen, S. Kim, W. Mu, S. K. Gazi,
R. K. Barrow, G. Yang, R. Wang and S. H. Snyder, Sci. Signaling, 2009,
2, ra72.
6
M. S. Vandiver, B. D. Paul, R. Xu, S. Karuppagounder, F. Rao, A. M.
Snowman, H. Seok Ko, Y. Il Lee, V. L. Dawson, T. M. Dawson, N. Sen
and S. H. Snyder, Nat. Commun., 2013, 4, 1626.
7
8
T. S. Bailey, L. N. Zakharov and M. D. Pluth, J. Am. Chem. Soc., 2014,
136, 10573–10576.
T. Ida, T. Sawa, H. Ihara, Y. Tsuchiya, Y. Watanabe, Y. Kumagai,
M. Suematsu, H. Motohashi, S. Fujii, T. Matsunaga, M. Yamamoto,
K. Ono, N. O. Devarie-Baez, M. Xian, J. M. Fukuto and T. Akaike,
Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 7606–7611.
Fig. 3 Bioluminescent response of (a) D/L-Cys with CBT-OH in the presence/
9
M. Ishigami, K. Hiraki, K. Umemura, Y. Ogasawara, K. Ishii and
H. Kimura, Antioxid. Redox Signaling, 2009, 11, 205–214.
absence of DAO and (b) D-Cys with the competitive inhibitor benzoate or
À1
potential substrate D-serine. Conditions: 20 mM Cys, 0.1 mg mL DAO, 40 mM
10 N. Shibuya, S. Koike, M. Tanaka, M. Ishigami-Yuasa, Y. Kimura,
FAD. 50 mM pH 7.4 tris buffer, 37 1C. Competition experiments were performed
Y. Ogasawara, K. Fukui, N. Nagahara and H. Kimura, Nat. Commun.,
with 2 mM sodium benzoate or 20 mM D-serine. After incubation, each sample
2013, 4, 1366.
À1
was quenched with 100 mM CBT-OH and imaged with 0.1 mg mL P. pyralis. at 11 L. Pollegioni, L. Piubelli, S. Sacchi, M. S. Pilone and G. Molla, Cell.
2+
5
60 nm for 45 min in 10 mM Mg , and 2 mM ATP.
Mol. Life Sci., 2007, 64, 1373–1394.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun.

View Article Online
Communication
ChemComm
1
2 G. Tedeschi, L. Pollegioni and A. Negri, in Unnatural Amino Acids, 16 G. C. Van de Bittner, C. R. Bertozzi and C. J. Chang, J. Am. Chem.
ed. L. Pollegioni and S. Servi, Humana Press, 2012, vol. 794,
pp. 381–395.
Soc., 2013, 135, 1783–1795.
17 H. Ren, F. Xiao, K. Zhan, Y.-P. Kim, H. Xie, Z. Xia and J. Rao, Angew.
Chem., 2009, 121, 9838–9842.
1
1
1
3 W. Wu, J. Li, L. Chen, Z. Ma, W. Zhang, Z. Liu, Y. Cheng, L. Du and
M. Li, Anal. Chem., 2014, 86, 9800–9806.
4 G. C. Van de Bittner, E. A. Dubikovskaya, C. R. Bertozzi and
C. J. Chang, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 21316–21321.
5 Z. Yao, H. Bai, C. Li and G. Shi, Chem. Commun., 2011, 47,
18 D. P. Nguyen, T. Elliott, M. Holt, T. W. Muir and J. W. Chin, J. Am.
Chem. Soc., 2011, 133, 11418–11421.
19 J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang,
I. A. Miller, A. Lo, J. A. Codelli and C. R. Bertozzi, Proc. Natl. Acad.
Sci. U. S. A., 2007, 104, 16793–16797.
7
431–7433.
Chem. Commun.
This journal is ©The Royal Society of Chemistry 2014