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Purification and characterization of theobromine synthase in a Theobromine-Enriched wild tea plant (Camellia gymnogyna Chang) from Dayao Mountain, China
Journal Pre-proofs Purification and Characterization of Theobromine Synthase in a TheobromineEnriched Wild Tea Plant (Camellia gymnogyna Chang) from Dayao Mountain, China Jie Teng, Changyu Yan, Wen Zeng, Yuqian Zhang, Zhen Zeng, Yahui Huang PII: DOI: Reference:
S0308-8146(19)32013-8 https://doi.org/10.1016/j.foodchem.2019.125875 FOCH 125875
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
11 June 2019 2 November 2019 6 November 2019
Please cite this article as: Teng, J., Yan, C., Zeng, W., Zhang, Y., Zeng, Z., Huang, Y., Purification and Characterization of Theobromine Synthase in a Theobromine-Enriched Wild Tea Plant (Camellia gymnogyna Chang) from Dayao Mountain, China, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem. 2019.125875
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1
Purification and Characterization of Theobromine Synthase in a
2
Theobromine-Enriched Wild Tea Plant (Camellia gymnogyna Chang)
3
from Dayao Mountain, China
4 5
Jie Tenga,b, Changyu Yana, Wen Zenga, Yuqian Zhanga, Zhen Zenga*, Yahui
6
Huanga,c*
7
a
8
University, Guangzhou 510642, China
9
b
Department of Tea Science, College of Horticulture, South China Agricultural
Department of Tea Science, School of Agricultural Sciences, Jiangxi Agricultural
10
University, Nanchang 330045, China
11
c
12
Guangzhou 510642, China
13
Corresponding Author
14
*Zhen
15
E-mail: [email protected];
16
*Yahui
17
E-mail: [email protected].
Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods,
Zeng, phone: +86 020 3829 7601, fax: +86 020 8528 0228,
Huang, phone: +86 020 3829 7601,
18 19 20 21 22 1
23
Abstract
24
Camellia gymnogyna Chang (CgC), a wild tea plant, was discovered on Dayao
25
Mountain, China. However, research regarding this tea plant is limited. Our study
26
found that CgC contains theobromine, caffeine, and theacrine, among which
27
theobromine content was the highest (14.37-39.72 mg/g). In addition, theobromine
28
synthase (TS) was partially purified from CgC leaves, up to 35.87-fold, with
29
consecutive chromatography, and its molecular weight was found to be approximately
30
62 kDa. The optimum reaction time, pH, and temperature for theobromine synthase
31
from 7-methylxanthine was found to be 6 h, 4, and 45 °C, respectively. TS expression
32
at both mRNA and protein stages was higher in the first than in the fourth leaf (P <
33
0.05). Subcellular localization of TS indicated that it was localized in the nucleus.
34
These results indicate that CgC can be of scientific value and could lead to efficient
35
utilization of this rare wild tea germplasm.
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Keywords: Purine alkaloid, Theobromine synthase, Purification, Wild tea, Dayao
37
Mountain
38 39 40 41 42 43 44 2
45
Abbreviations: CgC, Camellia gymnogyna Chang; TS, theobromine synthase; GFP,
46
green fluorescent protein; NMT, N-methyltransferase; XMT, xanthosine methy
47
transferase; MXMT, 7-methy xanthine methyl transferase; DXMT, 3,7-dimethyl
48
xanthine methyl transferase; SAM, S-adenosyl-L-methionine; TCS, tea caffeine
49
synthase; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis;
50
HPLC, high performance liquid chromatography; qRT-PCR, quantitative real-time
51
PCR; CNS, central nervous system.
52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 3
67
1. Introduction
68
Tea is one of the most widely consumed non-alcoholic beverages in the world,
69
possessing wide-ranging health benefits attributed to its numerous secondary
70
metabolites (Wei et al., 2018). The general concept of tea plants refers only to the
71
widely cultivated tea (Camellia Sinensis (L.) O. Kuntze) and Pu'er tea (Camellia.
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assamica (Mast.) Chang). However, in the botanical classification system, tea plants
73
encompass all species and varieties of the Camellia Sect. Thea (L.) Dyer (Jin et al.,
74
2018). Because tea trees are usually heterosexual plants, there are many variants
75
which have evolved from the primitive populations into multiple species, sub-species,
76
and variants during long-term phylogeny and genetic variation. The ecological types
77
are especially complicated. Tea originates from China, the principal tea-planting area
78
of the word. According to several studies reported in the literature (Chen et al., 2000;
79
Gong et al., 2006), tea plants (Camellia Sect. Thea (L.) Dyer) are divided into 5
80
categories, including Camellia tachangenensis F. C. Zhang, Camellia gymnogyna
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Chang, Camellia crassicolumna Chang, Camellia taliensis (W. W. Smith) Melehior
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and Camellia sinensis (L.) O. Kuntze. Currently, the tea being cultivated in tea
83
gardens is predominantly Camellia sinensis, while most wild tea trees are classified
84
into other categories.
85
Purine alkaloids, a group of metabolites commonly found in tea, coffee and cocoa
86
plants, are secondary metabolites derived from purine nucleotides. They include
87
caffeine, theobromine, theophylline, methyluric acid, xanthine, hypoxanthine,
88
paraxanthine and methyluric acid (Ashihara & Crozier, 1999) (Fig. S1). Camellia 4
89
sinensis contains abundant caffeine, but small amounts of theobromine and
90
theophylline (Wang et al., 2011). Cocoa tea (Camellia ptilophylla Chang), which
91
originated from Guangdong province, China, is a well-known naturally caffeine-free
92
tea plant containing predominantly theobromine (Li, Xing, Ng, Zhou, & Shi, 2018).
93
While theacrine was only reported in Kucha (Camellia assamica var. kucha) (Zheng,
94
Ye, Kato, Crozier, & Ashihara, 2002), and Camellia sinensis var. puanensis Kurihara.
95
(Li et al., 2017). In all tea plants, the biosynthesis and degradation of the purine
96
alkaloids can lead to transformation from one purine alkaloid to another, and caffeine
97
plays an important role in this process (Koyama, Tomoda, Kato, & Ashihara, 2003).
98
Methyl transfer is a critical steps in the synthesis of methylxanthine alkaloids, where
99
N-methyltransferases (NMTs) play a crucial role in the biosynthesis of purine
100
alkaloids (Uefuji, Ogita, Yamaguchi, Koizumi, & Sano, 2003). Depending on the
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nature of catalytic substrates and position of the methyl to be transferred, NMTs
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involved in caffeine biosynthesis are divided into xanthosine methyl transferase
103
(XMT)，7-methy xanthine methyl transferase (MXMT)，and 3,7-dimethyl xanthine
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methyl transferase (DXMT) (Ashihara et al., 2008; Cordell, 2013; Ogawa et al.,
105
2001). In a crude enzyme extract of tea leaves, purified NMTs and recombinant
106
protein expressed from the cloned NMT gene, the third and first methyl can be
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transferred in all cases, that is, their enzymatic properties are very similar. Therefore,
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these two enzymes are usually treated as the same enzyme known as tea caffeine
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synthase (TCS) (Uefuji, Ogita, Yamaguchi, Koizumi, & Sano, 2003). More
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importantly, there have been several reports on the extraction, isolation and 5
111
purification of NMT involved in the caffeine metabolism pathway, Mazzafera
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(Mazzafera, Wingsle, Olson, & Sandberg, 1994) first extracted NMT from coffee
113
endosperm and found that it catalyzed the conversion of 7-methylxanthine and
114
theobromine, but the activity of the isolated enzyme protein after purification was
115
extremely unstable. Subsequently, Waldhauser (Waldhauser, Gillies, Crozier, &
116
Baumann, 1997) partially purified an NMT by focused chromatography, and their
117
results showed enzymatic properties different from those involving two methyl
118
transfers. Kato (Kato et al., 1999) also purified an NMT relying on the substrate,
119
S-adenosyl-L-methionine (SAM) from fresh tea leaves; the enzyme protein exhibited
120
high substrate specificity at the third and the first methyltransferase, and the
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molecular weight was found to be approximately 41 kDa by SDS-PAGE
122
electrophoresis.
123
There is a myriad of wild type tea plant resources existing in the region of Dayao
124
Mountain, Guangxi Province, due to this area’s unique geology, diverse climate and
125
plentiful rainfall. Ordinarily, tea plant have to cross vast tracts of land from
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Yunan-Guizhou Plateau, Southwest China, leading to Southeast China. (Jiang et al.,
127
2017) The unique wild tea plant discovered in Dayao Mountain, CgC, is
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distinguishable from common tea plants by its purine alkaloid composition of three
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purines, namely theobromine, theacrine, and caffeine. Among which, the content of
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theobromine is the highest, while caffeine content is the lowest. Therefore, it is of
131
great scientific significance to study the factors contributing to this particular purine
132
alkaloid composition and its inherent characteristics. In this study, we characterized 6
133
the three purine alkaloids present in CgC, successfully purified and cloned TS from
134
CgC, and explored the properties of the key enzyme by subcellular localization,
135
qRT-PCR, and western-blot. Thus, this constitutes a systematic study of enzymatic
136
properties and the molecular mechanism of TS from CgC, a theobromine-enriched
137
wild tea plant growing in Guangxi, south China.
138
2. Materials and methods
139
2.1. Collection and pre-treatment of the tea samples
140
CgC specimens were collected their original growing regions, Dayao Mountain
141
(Guangxi Province, China) (Fig. S2). CgC is a kind of wild tea plant characterized by
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glabrous leaves, branches, flowers, fruits, seeds and ovaries; it has spherical seeds,
143
three locules in the flower ovaries, stamens that terminate in two whorls, and the outer
144
stamens connect to form filaments. Biochemical compositions of a number of CgC
145
plants were analyzed in a pre-experiment, the results of which confirmed that there
146
was no significant difference in purine alkaloid content among different plants (P >
147
0.05).
148
One bud and the fourth leaves were plucked from the CgC specimens obtained in
149
May 2016. These tea leaves were randomly divided into two sections. One section
150
was used for chemical compositions analysis (samples were fixed in a microwave and
151
then adequately dried); the other was used for RNA and theobromine synthase
152
extractions (fresh tea leaves were stored at -80 °C).
153
2.2. Chemicals
154
Caffeine (Purity >98.0%), theobromine (Purity >98.0%), and theophylline 7
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(Purity >98.0%) standards were purchased from Sigma-Aldrich (St. Louis, MO,
156
USA). The theacrine standard (Purity >98.0%) was obtained from Better-in
157
Pharmaceutical Technology Co., Ltd. (Shanghai, China). An Enhanced BCA Protein
158
Assay Kit was purchased from Sangon Biotech Co., Ltd. (Shanghai, China).
159
Ultrafiltration membranes (15 KDa cutoff) were purchased from Merck Millipore Co.,
160
Ltd. (Billerica, MA, USA). Q-Sepharose Fast Flow, Sephadex G-75, and dialysis
161
tubing (8-12 kDa cutoff) were purchased from Pharmacia & Upjohn Co., Lid.
162
(Kalamazoo, MI, USA). Enzyme substrates, extraction chemicals, and buffers were
163
either chromatography grade (HPLC) or reagent grade (AR).
164
2.3. Analyses of purine alkaloids
165
Fresh tea leaf samples (bud, first leaf, second leaf, third leaf, fourth leaf, and one
166
bud and two leaves) were individually ground to a fine powder with a mortar and
167
pestle in the presence of liquid nitrogen. Each powder sample accurately weighed (0.5
168
g) and extracted with 10 mL of distilled water at 90 °C for 30 min with shaking. The
169
extracts were filtered through filter paper (0.45 m), and the solid residues were
170
re-extracted once as described above, the supernatants of the extracted solutions were
171
collected and filtered through a 0.22 μm membrane before analysis.
172
High-performance liquid chromatography (HPLC) conditions were similar to those
173
described in our previous paper. (Teng, Zeng, & Huang, 2018) HPLC (Agilent 1200,
174
Agilent Technologies, Santa Clara, CA, USA), and C18 reverse-phase column (Hy
175
Persil ODS2, 4.6 mm × 250 mm, 5 μL) (Thermo Fisher Scientific, USA) were used.
176
HPLC conditions were as follows: solvent A was ultra-pure water and solvent B was 8
177
100% methanol; the column temperature was 25 °C and the UV detection wavelength
178
was 232 nm; and sample injection volume was 10 μL, and the flow speed was 1.0
179
mL/min. The gradient elution profile was as follows: 10% B (v/v) at 0 to 50 min, up
180
to 70% B at 60 min. Peaks were identified by comparison of retention times with
181
corresponding standards.
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2.4. Purification of theobromine synthase
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2.4.1. Preparation of crude extracts
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Extraction and initial purification of TS from fresh leaves was conducted according
185
to a previously reported method with slight modifications (Kato et al., 1999; Teng et
186
al., 2017). Briefly, 50.0 g of fresh leaves (one bud and two leaves) was homogenized
187
with cold Tris-HCl buffer (500 mL, 50 mmol/L, pH 7.3) containing 8% glycerol,
188
0.5% vitamin C, 5 mmol/L EDTA, and 6% PVPP , using a blender, and then stored at
189
4 °C for 6 h before being centrifuged at 15,000 ×g for 30 min at 4 °C. The pellet was
190
discarded, and (NH4)2SO4 was added to the crude extract at 50% saturated
191
concentration, and the resultant mixture was kept at 4 °C for 4 h. The mixture was
192
then centrifuged at 12,000 ×g for 15 min at 4 °C. The pellet was discarded, and the
193
supernatant was brought to 80% (NH4)2SO4 saturation, followed by incubation on ice
194
water for 6 h and centrifugation at 12,000 ×g for 15 min at 4 °C. The resultant pellet
195
was homogenized in Tris-HCl buffer (10 mL, 50 mmol/L, pH 7.9) containing 5%
196
glycerol and passed through dialysis tubing (8-12 kDa cutoff) against the same buffer
197
at 4 °C for 12 h. The dialysate was centrifuged at 8,000 ×g for 10 min to remove
198
precipitated denatured protein, and concentrated to an appropriate volume with 9
199
polyethylene glycol 2000 prior to removal of the supernatant.
200
2.4.2. Q-sepharose fast flow strong ion exchange chromatography
201
The crude enzyme extract was separated by a Q-Sepharose Fast Flow column (16 ×
202
120 mm inner diameter). The column was equilibrated with Tris-HCl buffer (50
203
mmol/L, pH 7.9) containing 5% glycerol, and elution was carried out with NaCl
204
buffer solution in a linear gradient of 0 to 0.6 mol/L at a flow rate of 1 mL/min;
205
fractions were collected at 2 mL/tube with a fraction collector. The elution process
206
continued until no absorbance was detected at 280 nm. Protein concentration and TS
207
activities of each fraction were estimated according to procedures detailed in section
208
2.4.5, and fractions showing enzymatic activity were combined and concentrated by
209
ultrafiltration (15 kDa cutoff, Merck Millipore Co., Ltd., Billerica, MA, USA).
210
2.4.3. Sephadex G-75 gel filtration chromatography
211
The fractions from the previous step was loaded onto Sephadex G-75 column (16 ×
212
700 mm inner diameter) equilibrated with Tris-HCl buffer (20 mmol/L, pH 7.9)
213
containing 5% glycerol and 5 mmol/L NaCl, and the sample was eluted with the same
214
equilibration buffer mixture at 0.5 mL/min and collected at 2 mL/tube, and the active
215
fractions were combined and concentrated by ultrafiltration (15 kDa cutoff).
216
2.4.4. SDS-PAGE electrophoresis
217
Separation of enzymatic proteins according to their molecular weights was
218
achieved by SDS-PAGE using 12% (w/v) polyacrylamide gel, following the mothed
219
of Teng et al., (Teng, et al., 2017). The proteins were stained with Coomassie Brilliant
220
Blue R-250 using the method described by Liu et al. (Liu, Gao, Liu, Yang, Lu, Nie, et 10
221
al., 2012).
222
2.4.5. Enzymatic activity and protein concentration assays
223
TS enzymatic activity was determined by a slightly modified HPLC method (Li et
224
al., 2017). The reaction mixture consisted of xanthosine (180 μL, 5 mmol/L),
225
paraxanthine (5 mmol/L) as the substrate, and Tris-HCl (700 μL, 50 mmol/L, pH 7.9)
226
containing MgCl2 (1 mmol/L), NaCl (10 mmol/L), and DTT (0.1 mmol/L). 20 μL of
227
TS enzyme solution was added and incubated at 37 °C for 6 h. An enzyme extract
228
inactivated at 90 °C served as the negative control. The enzymatic activity (U) was
229
determined and expressed as the amount of enzyme consumed per minute in the
230
substrate solution (1 μmol/L), and taken as an enzyme activity unit. Protein content
231
was determined using the Enhanced BCA Protein Assay Kit (Sangon Biotech Co.,
232
Ltd., Shanghai, China) in accordance with the manufacturer's instructions. The
233
specific activity of TS was determined and expressed by enzyme activity per mg of
234
protein.
235
For HPLC analysis, solvent A was 0.2% acetic acid in water and solvent B was
236
acetonitrile; column temperature was 28 °C, and UV spectra were obtained at 274 nm.
237
After injection of the reaction mixture (10 μL), a linear gradient with a flow rate of
238
1.0 mL/min was established as follows: initial solvent ratio of 8% B was increased to
239
17% B (v/v) over 25 min and maintained for 5 min; then 17% to 90% B between 30
240
and 35 min, and from 90% to 8% B between 35 and 40 min. Peaks were identified by
241
comparison of their retention times with those of corresponding standards.
242
2.5. Properties of the purified theobromine synthase 11
243
2.5.1. Substrate specificity
244
To determine the Michaelis-Menten constant (Km), maximum velocity (Vmax), and
245
Kcat of TS reactions (Lineweaver & Burk, 1934), TS activity was measured with
246
substrates 7-methylxanthine, paraxanthine, xanthine, xanthosine, theobromine,
247
caffeine, and theophylline at concentrations of 0.1, 0.125, 0.15, 0.2, 0.25, 0.5, and 1.0
248
mmol/L, respectively. Reaction rates were determined according to the enzymatic
249
activity measurement method.
250
2.5.2. Reaction time and pH optima
251
To determine optimum reaction time, TS activity was measured with the most
252
suitable substrate at pH 7.9 and 50 mmol/L Tris-HCl buffer in reaction times ranging
253
from 1 h to 10 h. The same enzyme reacts differently under different pH conditions,
254
and the pH at which enzymatic activity is the highest was taken as the optimal pH for
255
that enzyme. For determination of the optimum pH for TS, phosphate buffer (pH
256
6.5-7.0) and Tris-HCl buffer (pH 7.5-9.0) were tested, while all other assay conditions
257
were unchanged, as per enzyme activity measurement parameters.
258
2.5.3. Optimal temperature and thermal stability
259
TS activity was determined using the optimal substrate in Tris-HCl buffer (50
260
mmol/L, pH 7.5) at 25-55 °C, with all other assay conditions unchanged as per
261
enzymatic activity measurement parameters. To test the relationship between thermal
262
stability and TS activity, the enzyme was incubated at 30-80 °C for 20 min prior to
263
the enzymatic activity assay. Enzymatic activity was then recorded at optimal
264
conditions for each reaction temperature in order to determine the temperature at 12
265
which the enzyme became heat-inactivated.
266
2.6. Total RNA extraction and cDNA cloning of TS
267
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA)
268
according to the manufacturer’s instructions. A total of 1.5 μg RNA was used to
269
synthesize the first strand of cDNA using an RNA PCR Kit Ver. 2.1 (Takara, Japan).
270
According to the conserved sequence (http://www.ncbi.nlm.nih.gov/pubmed/),
271
reverse transcription was designed for the TS gene using Primer 5 software (Premier
272
Co., Canada). The forward primer was ATGGAGCTAGCTACTGCG, and the reverse
273
primer was CTATCCATCAATCTTGGAAAGCAC. The protocol used was as
274
follows: 94 °C held for 2 min; then 35 cycles of the following sequence: 30 s at 94 °C,
275
30 s at 56 °C, and 1 min at 68 °C, for the purpose of amplification. PCR products
276
were analyzed on a 1% (w/v) agarose gel, and DNA was digested with BamH I and
277
Hind III to afford the expected fragment. The fragment was recovered from the
278
agarose gel using a Qiaquick gel extraction kit (QIAGEN, Germany) and cloned into
279
a pMD20-T Vector (Qiagen, Tokyo) according to the manufacturer’s instructions.
280
2.7. Phylogenetic analysis
281
To investigate evolutionary relationships between TS and related enzymes, a
282
multiple alignment of NMT members from various tea plants and others plants was
283
constructed with Clustal W 1.81. Based on the alignment, an un-rooted molecular
284
phylogenetic tree was constructed by the neighbor-joining method with bootstrap
285
analysis and Kimura’s correction for protein distances (Ahn, Saino, Mizutani,
286
Shimizu, & Sakata, 2007). 13
287
2.8. Transient expression of GFP fusions
288
Green fluorescent protein (GFP) was used as a reporter protein in order to examine
289
the subcellular localization of TS. The TS cDNA fragment was amplified by PCR
290
using oligonucleotides containing restriction sites: TS-Sal I 5’-ACTGGTACCCGGG
291
GATCCATGGGGAAGGTGAACGAA-3’ and TS-BamH I 5’-CTTGCTCACCATGT
292
CGACTCCAACAATCTTGGAAAG-3’. The amplified DNA fragment was ligated
293
into the pC1301 vector (Promega, Madison, WI, USA) and digested with Sal I and
294
BamH I. The constructed pC1301-GFP-TS targeting plasmid was added to 20 μL of
295
EHA105 Agrobacterium competent cells, mixed well, and cooled in an ice-bath for 30
296
min. Then, 975 μL of antibiotic-free Agrobacterium medium was added, and the
297
mixture was shocked with a Gene Pulser Xcell transformer (Bio-rad, Hercules, CA,
298
USA) for fusion. After bombardment, cells were incubated for 3 h at 28 °C in a solid
299
MS medium supplemented with kanamycin and rifampicin. When the OD600 for target
300
plasmid Agrobacterium reached a value of approximately 0.3, it was suspended in an
301
equal volume of buffer (10 mmol/L 2-morpholino-ethanesulfonic acid, 200 μmol/L
302
acetosyringone, and 10 mmol/L MgCl2) and left at room temperature for 4-6 h, and
303
then injected into epidermal tissue of N. benthamiana tobacco leaves. Fluorescence
304
was observed using confocal laser-scanning microscopy 4-6 days later.
305
2.9. Quantitative real-time PCR analysis
306
Total RNA was extracted from different leaf positions (the first and the fourth leaf).
307
Quantitative real-time PCR (qRT-PCR) was performed using 2 μL of cDNA and 0.4
308
μmol/L of each primer in a 20 μL reaction volume with SYBR Premix Ex TaqII 14
309
(Takara, Japan). GAPDH was used as an internal control, and the specific primers
310
used for qRT-PCR analysis were designed based on the open reading frame (ORF) of
311
TS
312
CTATCCATCA ATCTTGGAAAGCAC). Each PCR was performed in three
313
replicates, and the expression level in the first leaf was taken as the control.
314
According to the threshold cycle (Ct), the changes in gene expression level were
315
quantified using the 2–ΔΔCt method (Livak & Schmittgen, 2001).
316
2.10.
(Forward
primer:
ATGGAGCTAGCTACTGCG,
Reverse
primer:
Preparation of TS polyclonal antibodies
317
This experiment was conducted as previously described (Mizutani et al., 2002) with
318
slight modifications. The cDNA encoding for the mature form prepared above was
319
inserted into the pMD20-T Vector (Qiagen, Tokyo). E. coli cells (DE3) were
320
transformed with the expression vector and the transformed cells were subsequently
321
grown in LB solid medium containing 50 μg/mL ampicillin, overnight at 37 °C with
322
shaking at 180 r/min. This overnight culture (1 mL) was used to inoculate LB liquid
323
medium (250 mL), supplemented with 50 μg/mL ampicillin and 25 μg/mL
324
clarithromycin. The cells were grown at 37 °C with shaking at 180 r/min, until OD600
325
reached approximately 0.4~0.6, the expression of TS fused with 6 × His-tag was
326
induced by the addition of 1 mmol/L IPTG at 37 °C for 24 h. The cells were collected
327
by centrifugation at 8,000 ×g for 15 min, re-suspended in sodium phosphate buffer
328
(0.1 mol/L, pH 7.8) containing 500 mmol/L NaCl, disrupted 20 times by sonication
329
(ice water) for 30 s with 15 s intervals, and centrifuged at 11,000 ×g for 10 min.
330
Because the expressed protein was insoluble and obtained as inclusion bodies, the 15
331
precipitates were dissolved in sodium phosphate buffer (0.1 mol/L, pH 7.8) containing
332
8.0 mol/L urea. The His tag fusion protein was purified according to the
333
manufacturer’s instructions.
334
Polyclonal antibodies against the purified recombinant protein were prepared by
335
HuaAn Biotechnology Co., Ltd. (Hangzhou, Zhejiang, China) in white rabbits by
336
standard methods. IgG in blood serum was purified on a Protein A Sepharose Column
337
(LIANKE Biotech, Co., Ltd., Hangzhou, Zhejiang, China) according to the
338
manufacturer’s instructions.
339
2.11.
Western blot
340
Tea shoots were separated into two sections (the first leaf and the fourth leaves),
341
and the total protein extraction was performed according to the crude enzyme
342
preparation method. A moderate amount of protein (20 g) from the crude extract was
343
loaded onto 12% (w/v) SDS-PAGE, and transferred to 0.45 μm polyvinylidene
344
fluoride membrane (PVDF) with a transfer apparatus (Bio-Rad, USA). β-actin was
345
taken as an internal control for total protein loading and the bound anti-TS antibody
346
was detected using a goat anti-rabbit IgG conjugated to alkaline phosphatase and
347
Western Blotting Kit (Sangon Biotech Co., Ltd., Shanghai, China).
348
2.12.
Statistical analysis
349
All data were subjected to analysis of variance using SPSS (19.0, SPSS Inc.,
350
Chicago, IL, USA). The statistical analyses were performed using one-way analysis of
351
variance (ANOVA), followed by least significant difference (LSD) test and Duncan’s
352
test. 16
353
3. Results
354
3.1. Purine alkaloid content
355
As shown in Table 1, CgC from Dayao Mountain contained three purine alkaloids,
356
namely, theobromine, caffeine and theacrine. Theobromine content was the highest,
357
ranging from 14.37 to 39.72 mg/g, followed by that of theacrine, ranging from 5.82 to
358
9.10 mg/g, while caffeine content was the lowest, approximately 0.51-2.02 mg/g.
359
Using two leaves and one bud stage as a control (generally the picking standard for
360
tea processing), theobromine content was 5 times that of theacrine and 16 times that
361
of caffeine. By comparing the same leaf position in plants of varying maturity, it was
362
found that the contents of theobromine and caffeine gradually decreased with life
363
maturity, and their content in the first leaf was the highest; theacrine content in
364
different leaf positions did not vary significantly and increased slightly with maturity.
365
According to literature reports, Camellia sinensis contains only caffeine and
366
theobromine, with the caffeine content being the highest. Evidently, purine alkaloid
367
composition and content ratios of CgC are distinct from those of Camellia sinensis.
368
3.2. Partial purification of TS from Camellia gymnogyna Chang leaves
369
Q-sepharose Fast Flow chromatographic separation and purification results of TS
370
from CgC leaves are shown in Fig. 1A. A penetration peak appears during
371
equilibration in the initial 30 min, followed by a constant elution of substances during
372
the linear gradient elution period of 30-180 min, characterized by three distinct peaks.
373
Individual analysis of TS enzymatic activity for each collection tube revealed activity
374
in collection tubes No.40 to 46, with tube No. 43 showing the highest activity. 17
375
Enzymatically active fractions were combined and concentrated, and the reside
376
separated by Sephadex G-75 gel chromatography. The results are shown in Fig. 1B; a
377
single absorption peak appeared during the entire elution process, that is, between
378
tubes No. 66 to No. 89. Maximum absorption at 280 nm was detected for tube No. 70,
379
however, TS enzymatic activity was only detected in the eluate of tubes No. 70-74,
380
with No. 72 showing the highest activity. Eluates displaying TS enzymatic activity
381
were collected, concentrated and desalted.
382
Table 2 summarizes TS purification procedures, starting with 50 g of CgC leaves,
383
resulting in a 35.87-fold purification of TS, in a yield of 1.16%. Following Sephadex
384
G-75 chromatography, the purity of the preparation was assessed by SDS-PAGE
385
electrophoresis (Fig. 1C). The protein fractions contained one major protein
386
component with a molecular mass of 62 kDa, in addition to several minor
387
contaminants of approximately 70 kDa and 50 kDa. Due to limitations in plant
388
materials and potential loss of enzymatic activity through manipulation, we did not
389
perform further purification. Subsequently, peptide sequences of the TS enzyme could
390
not be obtained by mass spectrometry. Therefore, in order to identify the TS protein
391
during purification, we performed western blot analysis using an anti-TS polyclonal
392
antibody, where a single clear band was detected, identical to that of the dominant
393
protein in the solution with a molecular mass of 62 kDa (Fig. 1D).
394
3.3. Kinetic properties
395
Activities of the TS enzyme were examined in the presence of theobromine,
396
xanthosine, 7-dimethylxanthine, and caffeine as the substrates. As shown in Table 3, 18
397
reaction affinities of different substrates with the purified TS enzyme showed that the
398
Michaelis constant Km value varied from 41 to 197 μmol/L, with the order of substrate
399
binding affinity being 7-methylxanthine > xanthosine > xanthine > paraxanthine,
400
while theobromine, caffeine and theophylline didn’t react, indicating that
401
7-methylxanthine has a high affinity for theobromine synthase in CgC from Dayao
402
Mountain. In summary, the effects of these methyl receptor concentrations on the
403
activity of theobromine synthase showed typical Michaelis-Menten-type kinetics,
404
with a novel feature being that theobromine, caffeine and theophylline could not be
405
used as substrates for the TS enzyme of CgC.
406
3.4. Effect of reaction time and pH value on TS activity
407
Reaction time is a crucial factor in enzymatic systems. Curtailed reaction times lead
408
to insufficient reactions, whereas exceedingly long reaction times result in low
409
efficiency and even product transformation or degradation. Therefore, optimizing
410
reaction time can effectively improve product formation and conversion efficiency.
411
Results shown in Fig. 2A indicate that the conversion product of purified theobromine
412
synthase gradually increased in concentration between 1.5 and 6 h, reached a
413
maximum value at 6 h, with minimal conversion to products taking place from 6 to 10
414
h (P > 0.05). Hence, the optimum reaction time for this system is 6 h. In addition,
415
enzymes, which are extremely sensitive to strong acids or bases, usually exhibit
416
catalytic activity only in the appropriate pH environment. Inappropriate pH
417
environments can affect enzymatic activity by changing the conformation of the
418
enzyme protein. The effect of pH on TS activity was determined over a pH range of 19
419
6.5-9.0 (Fig. 2B). A rapid increase in enzymatic activity was recorded with an
420
increase in pH from 6.5 to 8.0, followed by an immediate decreased in activity when
421
pH changed from 8.0 to 9.0, with approximately only 25.68% of the relative
422
enzymatic activity being retained. Therefore, the optimal reaction pH for theobromine
423
synthase of CgC was determined to be 8.0, indicating that a relatively alkaline
424
environment is favorable for the TS activity.
425
3.5. Optimum temperature and thermal stability of TS
426
The effect of different reaction temperatures on theobromine synthase activity is
427
shown in Fig. 2C. TS manifested the highest enzymatic activity at 35 ℃, with activity
428
increasing rapidly within a temperature range of 25-35 ℃. However, within the range
429
of 35-50 ℃, TS activity declined gradually, and relative enzyme activity was
430
approximately 20%, indicating that the enzyme is poorly resistant to high
431
temperatures; enzymatic activity was virtually non-existent temperatures exceeding
432
50 ℃. Meanwhile, thermal stability can indirectly reflect the level of structural
433
stability of an enzyme. In this experiment, purified TS enzyme was incubated at
434
different temperatures for 20 min, prior to measuring enzymatic activity, under
435
optimal reaction conditions. The maximum enzymatic activity value was taken as
436
100% relative enzymatic activity, and the effects of incubation temperatures on the
437
activity of theobromine synthase are shown in Fig. 2D. With an increase in incubation
438
temperature, the enzymatic activity tended to decreased, i.e., it was negatively
439
correlated with temperature. At 40 ℃, 88.4% relative enzyme activity was retained,
440
while 62.7% relative enzyme activity was retained at 50 ℃, indicating that the 20
441
enzyme protein is stable in the temperature range of 30-50 ℃. However, only 27.1%
442
relative enzymatic activity was retained at 60 ℃, and the TS enzyme was completely
443
denatured at 70 ℃.
444
3.6. Phylogenetic analysis
445
Phylogenetic analysis of the amino acid sequence encoding TS in Camellia
446
gymnogyna Chang (Fig. S3) was compared with other plant-derived amino acids
447
encoding for N-methyltransferase, using MEGALIGN software. Results of the
448
analysis are shown in Fig. 3A. Based on the analyses of various tea tree species,
449
including Theobroma cacao, N-methyltransferase could be broadly classified into
450
three categories: those present in Camellia ptilophylla (BAE79732.1), Camellia
451
irrawadiensis (BAE79729.1), and Camellia japonica (BAG84612.1). Theobroma
452
cacao (BAE79730.1) and CgC are classified into the same category; Camellia sinensis
453
(ABP98983.1), Camellia crassicolumna (ALP01720.1) and Camellia taliensis
454
(ALP01721.1) belong to the same class; and Camellia kissii (BAG84615.1) and
455
Camellia petelotii (BAG84617.1) are part of the same class. It was shown that high
456
affinity exists between the amino acid sequence encoding TS in CgC and the
457
therobromine synthase of Camellia ptilophylla and Camellia irrawadiensis.
458
3.7. Leaf position-specific expression of TS transcript
459
qRT-PCR and western-blot analyses were performed on enzymes examined for
460
activity in two different leaf positions, the first leaf and fourth leaves, to demonstrate
461
differences in TS expression levels. The expression levels of the TS gene at mRNA
462
and protein levels were consistent, both being higher in the first leaf than those in the 21
463
fourth leaf (P < 0.05) (Fig. 3B and C), and both have a positive correlation with the
464
theobromine content.
465
3.8. Subcellular localization of TS
466
When the distribution of green fluorescence in cells is observed under a laser
467
confocal microscope, an excessively long dark culture time will result in no
468
observation of green fluorescence or a decrease in light intensity which influences the
469
observation. Because green fluorescent protein (GFP) is transiently expressed, it
470
cannot be maintained for extended periods of time. Experimental results are shown in
471
Fig. 3D, the red light corresponds to chloroplast self-luminescence, and green
472
fluorescence was a result of GFP luminescence, which in the overlay was observed in
473
the nucleus, indicating that TS is localized in the nucleus.
474
4. Discussion
475
Ordinarily, the dry weight content of caffeine in Camellia sinensis is 2~4%, relative
476
content of theobromine is approximately 0.05%, and that of theophylline is
477
approximately 0.002%; theacrine is predominantly distributed in the young bud and
478
leaves of Camellia assamica var. kucha, with a relative content of approximately
479
0.3~3%. Purine alkaloids have a variety of physiological effects, however there are
480
significant differences between individual compounds. Caffeine is the principal
481
constituent substance affecting the taste of tea. Appropriate caffeine content is
482
beneficial to human health, however, high contents give rise to central nervous system
483
(CNS) excitability, resulting in poor sleep or excessive excitement (Roehrs & Roth,
484
2008). Theacrine can improve monoamine neurotransmitter disorders and protect 22
485
neurons in animals, thus, it can act as an anti-depressant and improve memory (Xie et
486
al., 2009). Variations in plant purine alkaloid compositions are primarily affected by
487
species, environment, climate and processing methods. According to previous
488
research, Camellia sinensis contains abundant caffeine but lower levels of
489
theobromine and theophylline. Camellia ptilophylla contains chiefly theobromine and
490
lower levels of caffeine, which contributes to its ability to reduce blood pressure and
491
lessen CNS excitability (Wu et al., 2014). The dominant purine alkaloid in a new
492
camellia species, Camellia assamica var. kucha, is theacrine (Yang, Ye, Xu, & Jiang,
493
2007). CgC is a novel and scarce tea resource, with purine alkaloid composition and
494
proportions that differ significantly from those found in the classes of tea trees
495
reported above. Therefore, it is of great scientific significance to uncover the causes of
496
such purine alkaloid compositions in CgC and their characteristics, as it would offer a
497
deeper insight into classes of tea trees, and importantly, into the metabolic pathways
498
of purine alkaloids.
499
Q-Sepharose Fast Flow strong anion exchange chromatography, Sephadex G-75 gel
500
chromatography and ultrafiltration membranes were used to isolate and purify the
501
theobromine synthase monomer. The low recovery and reduced activity of the
502
enzyme was mainly attributed to limited raw materials, the choice of chromatographic
503
methods and the poor stability of N-methyltransferase in vitro. Hence, isolated
504
N-methyltransferase possessed lower activity, and the purified theobromine synthase
505
amount was sufficient for purification at electrophoresis level. Based on the protein
506
standards, the molecular weight of theobromine synthase was estimated to be 23
507
approximately 62 kDa. Therefore, determination of the amino acid sequence, accurate
508
molecular weight, functional structure relationships and enzymatic properties of
509
theobromine synthase requires further exploration.
510
It is known from the purine alkaloid synthesis pathway (Fig. S1) that caffeine
511
biosynthesis can utilize pseudo-xanthine and theophylline as precursors in the salvage
512
pathway, in addition to the primary pathway with theobromine. In the experiment
513
with various substrate and the enzyme monomer, it was found that purified caffeine
514
from CgC could not participate as a TS enzyme substrate. This is in contrast to results
515
obtained in a substrate specificity study of N-methyltransferase of Camellia assamica
516
var. kucha reported by Zheng (Zheng, Ye, Kato, Crozier, & Ashihara, 2002), who
517
found that paraxanthine, 7-methylxanthine, theobromine and caffeine were utilized as
518
methyl donors in the reaction with theacrine synthase. We concluded that there are
519
alternative enzymatic pathways involved in theacrine synthase in CgC, that are
520
distinct from the methylation site and catalytic action of the TS enzyme in Camellia
521
sinensis, Camellia ptilophylla and Camellia assamica var. kucha. This indicated that
522
there are significant differences in the recognition of key sites between theobromine
523
synthases purified from different plants and their methyl receptors, i.e., their
524
substrates. Unfortunately, since the Camellia assamica var. kucha synthetic base
525
enzyme pathways and genes require further exploration, there is no way to tell if a
526
salvage pathway for caffeine production exists in CgC; or, whether altering the
527
contents of theobromine and caffeine would achieve regulation of theacrine content.
528
These questions require further studies in more detail. 24
529
5. Conclusion
530
This study
has verified that the purine alkaloids of CgC from Dayao Mountain
531
consisted of theobromine, caffeine and theacrine, and that theobromine content was
532
the highest, followed by that of theacrine, and the lowest being caffeine. This purine
533
alkaloid composition and ratio is clearly distinguishable from Camellia sinensis,
534
Camellia ptilophylla and Camellia assamica var. kucha. The leaching tissue
535
homogenate method, ammonium sulfate precipitation, dialysis, strong anion exchange
536
chromatography, gel filtration chromatography, and ultrafiltration were used to
537
partially purify theobromine synthase (TS) from CgC. The purified enzyme study in
538
which various substrates were tested showed that enzyme affinity was as follows:
539
7-methylxanthine > xanthosine > xanthine > paraxanthine. The Km values were in the
540
range of 41-197 μmol/L, however theobromine, caffeine and theophylline did not
541
participate as substrates in the enzymatic reaction. Moreover, the optimal reaction
542
time of theobromine synthase was 6 h, the optimum pH was 8.0, and the optimum
543
reaction temperature was 35 ℃. After treatment at 60 ℃ for 20 min, relative enzyme
544
activity was only 27.1%, and the enzyme became thoroughly inactivated with an
545
increase in temperature beyond 60 ℃. Subcellular localization experiments showed
546
that the TS gene was localized in the nucleus. In the qRT-PCR and Western-blot
547
protein expression experiments of the TS enzyme, regardless of whether it was at the
548
gene level or protein level, the expression level in the first leaves of the tea was higher
549
than that in the fourth leaves, that is, the expression in young leaves was higher than
550
that of the older leaves. Consequently, theobromine contents, TS gene expression and 25
551
TS protein expression were positively correlated with each other. This study provides
552
a unique perspective on N-methyltransferases in tea plants and will hopefully renew
553
interests into their further exploration.
554
Acknowledgement
555
We thank Drs. Jinqiang Chen for critical review of this manuscript, and we would
556
like to thank Editage (www.editage.cn) for English language editing. This work was
557
supported by the Fu Jian Province “2011Collaborative Innovation Center” Chinese
558
Oolong Tea Industry Innovation Center Special Project (J2015-75); Guangdong Tea
559
Industry Research System (2019LM1117).
560
Conflict of interest
561 562 563 564
The authors notify that are no conflicts of interest. Ethical approval This study does not involve any human or animal testing. Supplementary data
565
Fig. S1. Purine alkaloid synthesis and metabolic pathways.
566
Fig. S2. Morphological characteristic of Camellia gymnogyna Chang from Dayao
567 568 569
Mountain: plant type and leaves. Fig. S3. Nucleotide and predicted amino acid sequences of theobromine synthase in a wild tea plant (Camellia gymnogyna Chang) from Dayao Mountain, China.
570 571
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29
Figure captions Fig. 1. Isolation, purification and detection of purified TS in Camellia gymnogyna Chang from Dayao Mountain. (A) Q-Sepharose Fast Flow ion exchange chromatography elution profile of the TS enzyme. (B) Sephadex G-75 gel filtration of Q-Sepharose Fast Flow ion exchange eluent TS activity combined fractionation curve. (C) SDS-PAGE of TS purification processes (Marker, protein standards; Lane 1, Crude enzyme extract from CgC; Lane 2, 50~80% (NH4)2SO4 saturated precipitation fractionation; Lane 3, Elution of TS enzyme activity peak of Q-Sepharose Fast Flow ion exchange chromatography; Lane 4, Sephadex G-75 gel filtration of Q-Sepharose Fast Flow ion exchange eluent TS activity combined fractionation). (D) Western blotting profile of various TS preparations with an anti-theobromine synthase polyclonal antibody. Fig. 2. Properties of purified theobromine synthase. Effect of reaction time (A), pH (B), temperature (C) and thermal stability (D) on the relative activity of TS in CgC from Dayao Mountain. Fig. 3. Characteristics of the theobromine synthase gene from Camellia gymnogyna Chang (Dayao Mountain). (A) The neighbor-joining phylogenetic tree of N-methyltransferase members from various plants. TS mRNA (B) and protein (C) relative expression levels in different leaf positions of CgC. * means significant differences (P < 0.05). (D) Subcellular localization of the TS fluorescent protein produced by vector transient in N.benthamiana leaves (Bars = 19.00 μm).
30
List of chemical compounds (No.: FOODCHEM-D-19-03917) 1 Caffeine (PubChem CID:2519) 2 Theobromine (PubChem CID:5429) 3 Theophylline (PubChem CID:2153) 4 Theacrine (PubChem CID:75324) 5 7-methylxanthine (PubChem CID:68374) 6 Paraxanthine (PubChem CID:5687) 7 Xanthine (PubChem CID:1188) 8 Xanthosine (PubChem CID:64959)
TABLES Table 1. The contents of purine alkaloids in Camellia gymnogyna Chang from Dayao Mountain. Leaf positions
Camellia
Theobromine (mg/g)
Theacrine (mg/g)
Caffeine (mg/g)
Bud
34.45±1.37b
5.82±0.04a
1.73±0.05c
The first leaf
39.72±1.43a
6.33±0.05d
2.02±0.06a
The second leaf
23.82±1.29d
6.86±0.09c
1.86±0.03b
The third leaf
13.46±0.84e
7.36±0.12b
1.46±0.04d
The fourth leaf
14.37±0.99f
9.10±0.11a
0.51±0.02e
A bud and two leaf
31.57±1.61c
6.36±0.07d
1.94±0.05b
gymnogyna Chang
Note: Different small letters in same column data indicate significant difference at the P< 0.05 level.
31
Table 2. Summary of isolation and purification of the TS in Camellia gymnogyna Chang from Dayao Mountain. Volume
Enzymatic activity
Total protein
Specific activity
Purification
Yiel
(mL)
(U)
(mg)
(U/mg)
fold
(%)
384
1463
154.34
9.48
1.00
100
28
837
66.59
12.57
1.32
57.2
Q-Sepharose F F
14
91
1.20
75.83
8.00
6.22
Sephadex G-75
6
29
0.16
181.25
19.12
1.98
1.5
17
0.05
340.00
35.87
1.16
Step Crude enzyme 50~80% (NH4)2SO4 precipitate
Ultrafiltration
Table 3. Kinetic properties of TS in Camellia gymnogyna Chang from Dayao Mountain. NO.
Substrates
1
7-methylxanthine
Chemical formula
Km (μmol/L)
Vmax (U/mg)
Kcat (1/S)
41
237
301
2
Paraxanthine
197
65
82
3
Xanthine
144
89
112
32
4
Xanthosine
86
192
237
5
Theobromine
nd
nd
nd
6
Caffenine
nd
nd
nd
7
Theophylline
nd
nd
nd
nd, Not detected.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Highlights
Camellia gymnogyna Chang tea plant, found in Guangxi Province contains three different purine alkaloids. 33
Theobromine synthase was from Camellia gymnogyna Chang, purified, and characterized.
7-methylxanthine was a favorable substrate for theobromine synthase.
Full length cloning, subcellular localization, and polygenetic tree analysis of the theobromine synthase gene.
34
35
S0308-8146(19)32013-8 https://doi.org/10.1016/j.foodchem.2019.125875 FOCH 125875
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
11 June 2019 2 November 2019 6 November 2019
Please cite this article as: Teng, J., Yan, C., Zeng, W., Zhang, Y., Zeng, Z., Huang, Y., Purification and Characterization of Theobromine Synthase in a Theobromine-Enriched Wild Tea Plant (Camellia gymnogyna Chang) from Dayao Mountain, China, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem. 2019.125875
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© 2019 Published by Elsevier Ltd.
1
Purification and Characterization of Theobromine Synthase in a
2
Theobromine-Enriched Wild Tea Plant (Camellia gymnogyna Chang)
3
from Dayao Mountain, China
4 5
Jie Tenga,b, Changyu Yana, Wen Zenga, Yuqian Zhanga, Zhen Zenga*, Yahui
6
Huanga,c*
7
a
8
University, Guangzhou 510642, China
9
b
Department of Tea Science, College of Horticulture, South China Agricultural
Department of Tea Science, School of Agricultural Sciences, Jiangxi Agricultural
10
University, Nanchang 330045, China
11
c
12
Guangzhou 510642, China
13
Corresponding Author
14
*Zhen
15
E-mail: [email protected];
16
*Yahui
17
E-mail: [email protected].
Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods,
Zeng, phone: +86 020 3829 7601, fax: +86 020 8528 0228,
Huang, phone: +86 020 3829 7601,
18 19 20 21 22 1
23
Abstract
24
Camellia gymnogyna Chang (CgC), a wild tea plant, was discovered on Dayao
25
Mountain, China. However, research regarding this tea plant is limited. Our study
26
found that CgC contains theobromine, caffeine, and theacrine, among which
27
theobromine content was the highest (14.37-39.72 mg/g). In addition, theobromine
28
synthase (TS) was partially purified from CgC leaves, up to 35.87-fold, with
29
consecutive chromatography, and its molecular weight was found to be approximately
30
62 kDa. The optimum reaction time, pH, and temperature for theobromine synthase
31
from 7-methylxanthine was found to be 6 h, 4, and 45 °C, respectively. TS expression
32
at both mRNA and protein stages was higher in the first than in the fourth leaf (P <
33
0.05). Subcellular localization of TS indicated that it was localized in the nucleus.
34
These results indicate that CgC can be of scientific value and could lead to efficient
35
utilization of this rare wild tea germplasm.
36
Keywords: Purine alkaloid, Theobromine synthase, Purification, Wild tea, Dayao
37
Mountain
38 39 40 41 42 43 44 2
45
Abbreviations: CgC, Camellia gymnogyna Chang; TS, theobromine synthase; GFP,
46
green fluorescent protein; NMT, N-methyltransferase; XMT, xanthosine methy
47
transferase; MXMT, 7-methy xanthine methyl transferase; DXMT, 3,7-dimethyl
48
xanthine methyl transferase; SAM, S-adenosyl-L-methionine; TCS, tea caffeine
49
synthase; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis;
50
HPLC, high performance liquid chromatography; qRT-PCR, quantitative real-time
51
PCR; CNS, central nervous system.
52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 3
67
1. Introduction
68
Tea is one of the most widely consumed non-alcoholic beverages in the world,
69
possessing wide-ranging health benefits attributed to its numerous secondary
70
metabolites (Wei et al., 2018). The general concept of tea plants refers only to the
71
widely cultivated tea (Camellia Sinensis (L.) O. Kuntze) and Pu'er tea (Camellia.
72
assamica (Mast.) Chang). However, in the botanical classification system, tea plants
73
encompass all species and varieties of the Camellia Sect. Thea (L.) Dyer (Jin et al.,
74
2018). Because tea trees are usually heterosexual plants, there are many variants
75
which have evolved from the primitive populations into multiple species, sub-species,
76
and variants during long-term phylogeny and genetic variation. The ecological types
77
are especially complicated. Tea originates from China, the principal tea-planting area
78
of the word. According to several studies reported in the literature (Chen et al., 2000;
79
Gong et al., 2006), tea plants (Camellia Sect. Thea (L.) Dyer) are divided into 5
80
categories, including Camellia tachangenensis F. C. Zhang, Camellia gymnogyna
81
Chang, Camellia crassicolumna Chang, Camellia taliensis (W. W. Smith) Melehior
82
and Camellia sinensis (L.) O. Kuntze. Currently, the tea being cultivated in tea
83
gardens is predominantly Camellia sinensis, while most wild tea trees are classified
84
into other categories.
85
Purine alkaloids, a group of metabolites commonly found in tea, coffee and cocoa
86
plants, are secondary metabolites derived from purine nucleotides. They include
87
caffeine, theobromine, theophylline, methyluric acid, xanthine, hypoxanthine,
88
paraxanthine and methyluric acid (Ashihara & Crozier, 1999) (Fig. S1). Camellia 4
89
sinensis contains abundant caffeine, but small amounts of theobromine and
90
theophylline (Wang et al., 2011). Cocoa tea (Camellia ptilophylla Chang), which
91
originated from Guangdong province, China, is a well-known naturally caffeine-free
92
tea plant containing predominantly theobromine (Li, Xing, Ng, Zhou, & Shi, 2018).
93
While theacrine was only reported in Kucha (Camellia assamica var. kucha) (Zheng,
94
Ye, Kato, Crozier, & Ashihara, 2002), and Camellia sinensis var. puanensis Kurihara.
95
(Li et al., 2017). In all tea plants, the biosynthesis and degradation of the purine
96
alkaloids can lead to transformation from one purine alkaloid to another, and caffeine
97
plays an important role in this process (Koyama, Tomoda, Kato, & Ashihara, 2003).
98
Methyl transfer is a critical steps in the synthesis of methylxanthine alkaloids, where
99
N-methyltransferases (NMTs) play a crucial role in the biosynthesis of purine
100
alkaloids (Uefuji, Ogita, Yamaguchi, Koizumi, & Sano, 2003). Depending on the
101
nature of catalytic substrates and position of the methyl to be transferred, NMTs
102
involved in caffeine biosynthesis are divided into xanthosine methyl transferase
103
(XMT)，7-methy xanthine methyl transferase (MXMT)，and 3,7-dimethyl xanthine
104
methyl transferase (DXMT) (Ashihara et al., 2008; Cordell, 2013; Ogawa et al.,
105
2001). In a crude enzyme extract of tea leaves, purified NMTs and recombinant
106
protein expressed from the cloned NMT gene, the third and first methyl can be
107
transferred in all cases, that is, their enzymatic properties are very similar. Therefore,
108
these two enzymes are usually treated as the same enzyme known as tea caffeine
109
synthase (TCS) (Uefuji, Ogita, Yamaguchi, Koizumi, & Sano, 2003). More
110
importantly, there have been several reports on the extraction, isolation and 5
111
purification of NMT involved in the caffeine metabolism pathway, Mazzafera
112
(Mazzafera, Wingsle, Olson, & Sandberg, 1994) first extracted NMT from coffee
113
endosperm and found that it catalyzed the conversion of 7-methylxanthine and
114
theobromine, but the activity of the isolated enzyme protein after purification was
115
extremely unstable. Subsequently, Waldhauser (Waldhauser, Gillies, Crozier, &
116
Baumann, 1997) partially purified an NMT by focused chromatography, and their
117
results showed enzymatic properties different from those involving two methyl
118
transfers. Kato (Kato et al., 1999) also purified an NMT relying on the substrate,
119
S-adenosyl-L-methionine (SAM) from fresh tea leaves; the enzyme protein exhibited
120
high substrate specificity at the third and the first methyltransferase, and the
121
molecular weight was found to be approximately 41 kDa by SDS-PAGE
122
electrophoresis.
123
There is a myriad of wild type tea plant resources existing in the region of Dayao
124
Mountain, Guangxi Province, due to this area’s unique geology, diverse climate and
125
plentiful rainfall. Ordinarily, tea plant have to cross vast tracts of land from
126
Yunan-Guizhou Plateau, Southwest China, leading to Southeast China. (Jiang et al.,
127
2017) The unique wild tea plant discovered in Dayao Mountain, CgC, is
128
distinguishable from common tea plants by its purine alkaloid composition of three
129
purines, namely theobromine, theacrine, and caffeine. Among which, the content of
130
theobromine is the highest, while caffeine content is the lowest. Therefore, it is of
131
great scientific significance to study the factors contributing to this particular purine
132
alkaloid composition and its inherent characteristics. In this study, we characterized 6
133
the three purine alkaloids present in CgC, successfully purified and cloned TS from
134
CgC, and explored the properties of the key enzyme by subcellular localization,
135
qRT-PCR, and western-blot. Thus, this constitutes a systematic study of enzymatic
136
properties and the molecular mechanism of TS from CgC, a theobromine-enriched
137
wild tea plant growing in Guangxi, south China.
138
2. Materials and methods
139
2.1. Collection and pre-treatment of the tea samples
140
CgC specimens were collected their original growing regions, Dayao Mountain
141
(Guangxi Province, China) (Fig. S2). CgC is a kind of wild tea plant characterized by
142
glabrous leaves, branches, flowers, fruits, seeds and ovaries; it has spherical seeds,
143
three locules in the flower ovaries, stamens that terminate in two whorls, and the outer
144
stamens connect to form filaments. Biochemical compositions of a number of CgC
145
plants were analyzed in a pre-experiment, the results of which confirmed that there
146
was no significant difference in purine alkaloid content among different plants (P >
147
0.05).
148
One bud and the fourth leaves were plucked from the CgC specimens obtained in
149
May 2016. These tea leaves were randomly divided into two sections. One section
150
was used for chemical compositions analysis (samples were fixed in a microwave and
151
then adequately dried); the other was used for RNA and theobromine synthase
152
extractions (fresh tea leaves were stored at -80 °C).
153
2.2. Chemicals
154
Caffeine (Purity >98.0%), theobromine (Purity >98.0%), and theophylline 7
155
(Purity >98.0%) standards were purchased from Sigma-Aldrich (St. Louis, MO,
156
USA). The theacrine standard (Purity >98.0%) was obtained from Better-in
157
Pharmaceutical Technology Co., Ltd. (Shanghai, China). An Enhanced BCA Protein
158
Assay Kit was purchased from Sangon Biotech Co., Ltd. (Shanghai, China).
159
Ultrafiltration membranes (15 KDa cutoff) were purchased from Merck Millipore Co.,
160
Ltd. (Billerica, MA, USA). Q-Sepharose Fast Flow, Sephadex G-75, and dialysis
161
tubing (8-12 kDa cutoff) were purchased from Pharmacia & Upjohn Co., Lid.
162
(Kalamazoo, MI, USA). Enzyme substrates, extraction chemicals, and buffers were
163
either chromatography grade (HPLC) or reagent grade (AR).
164
2.3. Analyses of purine alkaloids
165
Fresh tea leaf samples (bud, first leaf, second leaf, third leaf, fourth leaf, and one
166
bud and two leaves) were individually ground to a fine powder with a mortar and
167
pestle in the presence of liquid nitrogen. Each powder sample accurately weighed (0.5
168
g) and extracted with 10 mL of distilled water at 90 °C for 30 min with shaking. The
169
extracts were filtered through filter paper (0.45 m), and the solid residues were
170
re-extracted once as described above, the supernatants of the extracted solutions were
171
collected and filtered through a 0.22 μm membrane before analysis.
172
High-performance liquid chromatography (HPLC) conditions were similar to those
173
described in our previous paper. (Teng, Zeng, & Huang, 2018) HPLC (Agilent 1200,
174
Agilent Technologies, Santa Clara, CA, USA), and C18 reverse-phase column (Hy
175
Persil ODS2, 4.6 mm × 250 mm, 5 μL) (Thermo Fisher Scientific, USA) were used.
176
HPLC conditions were as follows: solvent A was ultra-pure water and solvent B was 8
177
100% methanol; the column temperature was 25 °C and the UV detection wavelength
178
was 232 nm; and sample injection volume was 10 μL, and the flow speed was 1.0
179
mL/min. The gradient elution profile was as follows: 10% B (v/v) at 0 to 50 min, up
180
to 70% B at 60 min. Peaks were identified by comparison of retention times with
181
corresponding standards.
182
2.4. Purification of theobromine synthase
183
2.4.1. Preparation of crude extracts
184
Extraction and initial purification of TS from fresh leaves was conducted according
185
to a previously reported method with slight modifications (Kato et al., 1999; Teng et
186
al., 2017). Briefly, 50.0 g of fresh leaves (one bud and two leaves) was homogenized
187
with cold Tris-HCl buffer (500 mL, 50 mmol/L, pH 7.3) containing 8% glycerol,
188
0.5% vitamin C, 5 mmol/L EDTA, and 6% PVPP , using a blender, and then stored at
189
4 °C for 6 h before being centrifuged at 15,000 ×g for 30 min at 4 °C. The pellet was
190
discarded, and (NH4)2SO4 was added to the crude extract at 50% saturated
191
concentration, and the resultant mixture was kept at 4 °C for 4 h. The mixture was
192
then centrifuged at 12,000 ×g for 15 min at 4 °C. The pellet was discarded, and the
193
supernatant was brought to 80% (NH4)2SO4 saturation, followed by incubation on ice
194
water for 6 h and centrifugation at 12,000 ×g for 15 min at 4 °C. The resultant pellet
195
was homogenized in Tris-HCl buffer (10 mL, 50 mmol/L, pH 7.9) containing 5%
196
glycerol and passed through dialysis tubing (8-12 kDa cutoff) against the same buffer
197
at 4 °C for 12 h. The dialysate was centrifuged at 8,000 ×g for 10 min to remove
198
precipitated denatured protein, and concentrated to an appropriate volume with 9
199
polyethylene glycol 2000 prior to removal of the supernatant.
200
2.4.2. Q-sepharose fast flow strong ion exchange chromatography
201
The crude enzyme extract was separated by a Q-Sepharose Fast Flow column (16 ×
202
120 mm inner diameter). The column was equilibrated with Tris-HCl buffer (50
203
mmol/L, pH 7.9) containing 5% glycerol, and elution was carried out with NaCl
204
buffer solution in a linear gradient of 0 to 0.6 mol/L at a flow rate of 1 mL/min;
205
fractions were collected at 2 mL/tube with a fraction collector. The elution process
206
continued until no absorbance was detected at 280 nm. Protein concentration and TS
207
activities of each fraction were estimated according to procedures detailed in section
208
2.4.5, and fractions showing enzymatic activity were combined and concentrated by
209
ultrafiltration (15 kDa cutoff, Merck Millipore Co., Ltd., Billerica, MA, USA).
210
2.4.3. Sephadex G-75 gel filtration chromatography
211
The fractions from the previous step was loaded onto Sephadex G-75 column (16 ×
212
700 mm inner diameter) equilibrated with Tris-HCl buffer (20 mmol/L, pH 7.9)
213
containing 5% glycerol and 5 mmol/L NaCl, and the sample was eluted with the same
214
equilibration buffer mixture at 0.5 mL/min and collected at 2 mL/tube, and the active
215
fractions were combined and concentrated by ultrafiltration (15 kDa cutoff).
216
2.4.4. SDS-PAGE electrophoresis
217
Separation of enzymatic proteins according to their molecular weights was
218
achieved by SDS-PAGE using 12% (w/v) polyacrylamide gel, following the mothed
219
of Teng et al., (Teng, et al., 2017). The proteins were stained with Coomassie Brilliant
220
Blue R-250 using the method described by Liu et al. (Liu, Gao, Liu, Yang, Lu, Nie, et 10
221
al., 2012).
222
2.4.5. Enzymatic activity and protein concentration assays
223
TS enzymatic activity was determined by a slightly modified HPLC method (Li et
224
al., 2017). The reaction mixture consisted of xanthosine (180 μL, 5 mmol/L),
225
paraxanthine (5 mmol/L) as the substrate, and Tris-HCl (700 μL, 50 mmol/L, pH 7.9)
226
containing MgCl2 (1 mmol/L), NaCl (10 mmol/L), and DTT (0.1 mmol/L). 20 μL of
227
TS enzyme solution was added and incubated at 37 °C for 6 h. An enzyme extract
228
inactivated at 90 °C served as the negative control. The enzymatic activity (U) was
229
determined and expressed as the amount of enzyme consumed per minute in the
230
substrate solution (1 μmol/L), and taken as an enzyme activity unit. Protein content
231
was determined using the Enhanced BCA Protein Assay Kit (Sangon Biotech Co.,
232
Ltd., Shanghai, China) in accordance with the manufacturer's instructions. The
233
specific activity of TS was determined and expressed by enzyme activity per mg of
234
protein.
235
For HPLC analysis, solvent A was 0.2% acetic acid in water and solvent B was
236
acetonitrile; column temperature was 28 °C, and UV spectra were obtained at 274 nm.
237
After injection of the reaction mixture (10 μL), a linear gradient with a flow rate of
238
1.0 mL/min was established as follows: initial solvent ratio of 8% B was increased to
239
17% B (v/v) over 25 min and maintained for 5 min; then 17% to 90% B between 30
240
and 35 min, and from 90% to 8% B between 35 and 40 min. Peaks were identified by
241
comparison of their retention times with those of corresponding standards.
242
2.5. Properties of the purified theobromine synthase 11
243
2.5.1. Substrate specificity
244
To determine the Michaelis-Menten constant (Km), maximum velocity (Vmax), and
245
Kcat of TS reactions (Lineweaver & Burk, 1934), TS activity was measured with
246
substrates 7-methylxanthine, paraxanthine, xanthine, xanthosine, theobromine,
247
caffeine, and theophylline at concentrations of 0.1, 0.125, 0.15, 0.2, 0.25, 0.5, and 1.0
248
mmol/L, respectively. Reaction rates were determined according to the enzymatic
249
activity measurement method.
250
2.5.2. Reaction time and pH optima
251
To determine optimum reaction time, TS activity was measured with the most
252
suitable substrate at pH 7.9 and 50 mmol/L Tris-HCl buffer in reaction times ranging
253
from 1 h to 10 h. The same enzyme reacts differently under different pH conditions,
254
and the pH at which enzymatic activity is the highest was taken as the optimal pH for
255
that enzyme. For determination of the optimum pH for TS, phosphate buffer (pH
256
6.5-7.0) and Tris-HCl buffer (pH 7.5-9.0) were tested, while all other assay conditions
257
were unchanged, as per enzyme activity measurement parameters.
258
2.5.3. Optimal temperature and thermal stability
259
TS activity was determined using the optimal substrate in Tris-HCl buffer (50
260
mmol/L, pH 7.5) at 25-55 °C, with all other assay conditions unchanged as per
261
enzymatic activity measurement parameters. To test the relationship between thermal
262
stability and TS activity, the enzyme was incubated at 30-80 °C for 20 min prior to
263
the enzymatic activity assay. Enzymatic activity was then recorded at optimal
264
conditions for each reaction temperature in order to determine the temperature at 12
265
which the enzyme became heat-inactivated.
266
2.6. Total RNA extraction and cDNA cloning of TS
267
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA)
268
according to the manufacturer’s instructions. A total of 1.5 μg RNA was used to
269
synthesize the first strand of cDNA using an RNA PCR Kit Ver. 2.1 (Takara, Japan).
270
According to the conserved sequence (http://www.ncbi.nlm.nih.gov/pubmed/),
271
reverse transcription was designed for the TS gene using Primer 5 software (Premier
272
Co., Canada). The forward primer was ATGGAGCTAGCTACTGCG, and the reverse
273
primer was CTATCCATCAATCTTGGAAAGCAC. The protocol used was as
274
follows: 94 °C held for 2 min; then 35 cycles of the following sequence: 30 s at 94 °C,
275
30 s at 56 °C, and 1 min at 68 °C, for the purpose of amplification. PCR products
276
were analyzed on a 1% (w/v) agarose gel, and DNA was digested with BamH I and
277
Hind III to afford the expected fragment. The fragment was recovered from the
278
agarose gel using a Qiaquick gel extraction kit (QIAGEN, Germany) and cloned into
279
a pMD20-T Vector (Qiagen, Tokyo) according to the manufacturer’s instructions.
280
2.7. Phylogenetic analysis
281
To investigate evolutionary relationships between TS and related enzymes, a
282
multiple alignment of NMT members from various tea plants and others plants was
283
constructed with Clustal W 1.81. Based on the alignment, an un-rooted molecular
284
phylogenetic tree was constructed by the neighbor-joining method with bootstrap
285
analysis and Kimura’s correction for protein distances (Ahn, Saino, Mizutani,
286
Shimizu, & Sakata, 2007). 13
287
2.8. Transient expression of GFP fusions
288
Green fluorescent protein (GFP) was used as a reporter protein in order to examine
289
the subcellular localization of TS. The TS cDNA fragment was amplified by PCR
290
using oligonucleotides containing restriction sites: TS-Sal I 5’-ACTGGTACCCGGG
291
GATCCATGGGGAAGGTGAACGAA-3’ and TS-BamH I 5’-CTTGCTCACCATGT
292
CGACTCCAACAATCTTGGAAAG-3’. The amplified DNA fragment was ligated
293
into the pC1301 vector (Promega, Madison, WI, USA) and digested with Sal I and
294
BamH I. The constructed pC1301-GFP-TS targeting plasmid was added to 20 μL of
295
EHA105 Agrobacterium competent cells, mixed well, and cooled in an ice-bath for 30
296
min. Then, 975 μL of antibiotic-free Agrobacterium medium was added, and the
297
mixture was shocked with a Gene Pulser Xcell transformer (Bio-rad, Hercules, CA,
298
USA) for fusion. After bombardment, cells were incubated for 3 h at 28 °C in a solid
299
MS medium supplemented with kanamycin and rifampicin. When the OD600 for target
300
plasmid Agrobacterium reached a value of approximately 0.3, it was suspended in an
301
equal volume of buffer (10 mmol/L 2-morpholino-ethanesulfonic acid, 200 μmol/L
302
acetosyringone, and 10 mmol/L MgCl2) and left at room temperature for 4-6 h, and
303
then injected into epidermal tissue of N. benthamiana tobacco leaves. Fluorescence
304
was observed using confocal laser-scanning microscopy 4-6 days later.
305
2.9. Quantitative real-time PCR analysis
306
Total RNA was extracted from different leaf positions (the first and the fourth leaf).
307
Quantitative real-time PCR (qRT-PCR) was performed using 2 μL of cDNA and 0.4
308
μmol/L of each primer in a 20 μL reaction volume with SYBR Premix Ex TaqII 14
309
(Takara, Japan). GAPDH was used as an internal control, and the specific primers
310
used for qRT-PCR analysis were designed based on the open reading frame (ORF) of
311
TS
312
CTATCCATCA ATCTTGGAAAGCAC). Each PCR was performed in three
313
replicates, and the expression level in the first leaf was taken as the control.
314
According to the threshold cycle (Ct), the changes in gene expression level were
315
quantified using the 2–ΔΔCt method (Livak & Schmittgen, 2001).
316
2.10.
(Forward
primer:
ATGGAGCTAGCTACTGCG,
Reverse
primer:
Preparation of TS polyclonal antibodies
317
This experiment was conducted as previously described (Mizutani et al., 2002) with
318
slight modifications. The cDNA encoding for the mature form prepared above was
319
inserted into the pMD20-T Vector (Qiagen, Tokyo). E. coli cells (DE3) were
320
transformed with the expression vector and the transformed cells were subsequently
321
grown in LB solid medium containing 50 μg/mL ampicillin, overnight at 37 °C with
322
shaking at 180 r/min. This overnight culture (1 mL) was used to inoculate LB liquid
323
medium (250 mL), supplemented with 50 μg/mL ampicillin and 25 μg/mL
324
clarithromycin. The cells were grown at 37 °C with shaking at 180 r/min, until OD600
325
reached approximately 0.4~0.6, the expression of TS fused with 6 × His-tag was
326
induced by the addition of 1 mmol/L IPTG at 37 °C for 24 h. The cells were collected
327
by centrifugation at 8,000 ×g for 15 min, re-suspended in sodium phosphate buffer
328
(0.1 mol/L, pH 7.8) containing 500 mmol/L NaCl, disrupted 20 times by sonication
329
(ice water) for 30 s with 15 s intervals, and centrifuged at 11,000 ×g for 10 min.
330
Because the expressed protein was insoluble and obtained as inclusion bodies, the 15
331
precipitates were dissolved in sodium phosphate buffer (0.1 mol/L, pH 7.8) containing
332
8.0 mol/L urea. The His tag fusion protein was purified according to the
333
manufacturer’s instructions.
334
Polyclonal antibodies against the purified recombinant protein were prepared by
335
HuaAn Biotechnology Co., Ltd. (Hangzhou, Zhejiang, China) in white rabbits by
336
standard methods. IgG in blood serum was purified on a Protein A Sepharose Column
337
(LIANKE Biotech, Co., Ltd., Hangzhou, Zhejiang, China) according to the
338
manufacturer’s instructions.
339
2.11.
Western blot
340
Tea shoots were separated into two sections (the first leaf and the fourth leaves),
341
and the total protein extraction was performed according to the crude enzyme
342
preparation method. A moderate amount of protein (20 g) from the crude extract was
343
loaded onto 12% (w/v) SDS-PAGE, and transferred to 0.45 μm polyvinylidene
344
fluoride membrane (PVDF) with a transfer apparatus (Bio-Rad, USA). β-actin was
345
taken as an internal control for total protein loading and the bound anti-TS antibody
346
was detected using a goat anti-rabbit IgG conjugated to alkaline phosphatase and
347
Western Blotting Kit (Sangon Biotech Co., Ltd., Shanghai, China).
348
2.12.
Statistical analysis
349
All data were subjected to analysis of variance using SPSS (19.0, SPSS Inc.,
350
Chicago, IL, USA). The statistical analyses were performed using one-way analysis of
351
variance (ANOVA), followed by least significant difference (LSD) test and Duncan’s
352
test. 16
353
3. Results
354
3.1. Purine alkaloid content
355
As shown in Table 1, CgC from Dayao Mountain contained three purine alkaloids,
356
namely, theobromine, caffeine and theacrine. Theobromine content was the highest,
357
ranging from 14.37 to 39.72 mg/g, followed by that of theacrine, ranging from 5.82 to
358
9.10 mg/g, while caffeine content was the lowest, approximately 0.51-2.02 mg/g.
359
Using two leaves and one bud stage as a control (generally the picking standard for
360
tea processing), theobromine content was 5 times that of theacrine and 16 times that
361
of caffeine. By comparing the same leaf position in plants of varying maturity, it was
362
found that the contents of theobromine and caffeine gradually decreased with life
363
maturity, and their content in the first leaf was the highest; theacrine content in
364
different leaf positions did not vary significantly and increased slightly with maturity.
365
According to literature reports, Camellia sinensis contains only caffeine and
366
theobromine, with the caffeine content being the highest. Evidently, purine alkaloid
367
composition and content ratios of CgC are distinct from those of Camellia sinensis.
368
3.2. Partial purification of TS from Camellia gymnogyna Chang leaves
369
Q-sepharose Fast Flow chromatographic separation and purification results of TS
370
from CgC leaves are shown in Fig. 1A. A penetration peak appears during
371
equilibration in the initial 30 min, followed by a constant elution of substances during
372
the linear gradient elution period of 30-180 min, characterized by three distinct peaks.
373
Individual analysis of TS enzymatic activity for each collection tube revealed activity
374
in collection tubes No.40 to 46, with tube No. 43 showing the highest activity. 17
375
Enzymatically active fractions were combined and concentrated, and the reside
376
separated by Sephadex G-75 gel chromatography. The results are shown in Fig. 1B; a
377
single absorption peak appeared during the entire elution process, that is, between
378
tubes No. 66 to No. 89. Maximum absorption at 280 nm was detected for tube No. 70,
379
however, TS enzymatic activity was only detected in the eluate of tubes No. 70-74,
380
with No. 72 showing the highest activity. Eluates displaying TS enzymatic activity
381
were collected, concentrated and desalted.
382
Table 2 summarizes TS purification procedures, starting with 50 g of CgC leaves,
383
resulting in a 35.87-fold purification of TS, in a yield of 1.16%. Following Sephadex
384
G-75 chromatography, the purity of the preparation was assessed by SDS-PAGE
385
electrophoresis (Fig. 1C). The protein fractions contained one major protein
386
component with a molecular mass of 62 kDa, in addition to several minor
387
contaminants of approximately 70 kDa and 50 kDa. Due to limitations in plant
388
materials and potential loss of enzymatic activity through manipulation, we did not
389
perform further purification. Subsequently, peptide sequences of the TS enzyme could
390
not be obtained by mass spectrometry. Therefore, in order to identify the TS protein
391
during purification, we performed western blot analysis using an anti-TS polyclonal
392
antibody, where a single clear band was detected, identical to that of the dominant
393
protein in the solution with a molecular mass of 62 kDa (Fig. 1D).
394
3.3. Kinetic properties
395
Activities of the TS enzyme were examined in the presence of theobromine,
396
xanthosine, 7-dimethylxanthine, and caffeine as the substrates. As shown in Table 3, 18
397
reaction affinities of different substrates with the purified TS enzyme showed that the
398
Michaelis constant Km value varied from 41 to 197 μmol/L, with the order of substrate
399
binding affinity being 7-methylxanthine > xanthosine > xanthine > paraxanthine,
400
while theobromine, caffeine and theophylline didn’t react, indicating that
401
7-methylxanthine has a high affinity for theobromine synthase in CgC from Dayao
402
Mountain. In summary, the effects of these methyl receptor concentrations on the
403
activity of theobromine synthase showed typical Michaelis-Menten-type kinetics,
404
with a novel feature being that theobromine, caffeine and theophylline could not be
405
used as substrates for the TS enzyme of CgC.
406
3.4. Effect of reaction time and pH value on TS activity
407
Reaction time is a crucial factor in enzymatic systems. Curtailed reaction times lead
408
to insufficient reactions, whereas exceedingly long reaction times result in low
409
efficiency and even product transformation or degradation. Therefore, optimizing
410
reaction time can effectively improve product formation and conversion efficiency.
411
Results shown in Fig. 2A indicate that the conversion product of purified theobromine
412
synthase gradually increased in concentration between 1.5 and 6 h, reached a
413
maximum value at 6 h, with minimal conversion to products taking place from 6 to 10
414
h (P > 0.05). Hence, the optimum reaction time for this system is 6 h. In addition,
415
enzymes, which are extremely sensitive to strong acids or bases, usually exhibit
416
catalytic activity only in the appropriate pH environment. Inappropriate pH
417
environments can affect enzymatic activity by changing the conformation of the
418
enzyme protein. The effect of pH on TS activity was determined over a pH range of 19
419
6.5-9.0 (Fig. 2B). A rapid increase in enzymatic activity was recorded with an
420
increase in pH from 6.5 to 8.0, followed by an immediate decreased in activity when
421
pH changed from 8.0 to 9.0, with approximately only 25.68% of the relative
422
enzymatic activity being retained. Therefore, the optimal reaction pH for theobromine
423
synthase of CgC was determined to be 8.0, indicating that a relatively alkaline
424
environment is favorable for the TS activity.
425
3.5. Optimum temperature and thermal stability of TS
426
The effect of different reaction temperatures on theobromine synthase activity is
427
shown in Fig. 2C. TS manifested the highest enzymatic activity at 35 ℃, with activity
428
increasing rapidly within a temperature range of 25-35 ℃. However, within the range
429
of 35-50 ℃, TS activity declined gradually, and relative enzyme activity was
430
approximately 20%, indicating that the enzyme is poorly resistant to high
431
temperatures; enzymatic activity was virtually non-existent temperatures exceeding
432
50 ℃. Meanwhile, thermal stability can indirectly reflect the level of structural
433
stability of an enzyme. In this experiment, purified TS enzyme was incubated at
434
different temperatures for 20 min, prior to measuring enzymatic activity, under
435
optimal reaction conditions. The maximum enzymatic activity value was taken as
436
100% relative enzymatic activity, and the effects of incubation temperatures on the
437
activity of theobromine synthase are shown in Fig. 2D. With an increase in incubation
438
temperature, the enzymatic activity tended to decreased, i.e., it was negatively
439
correlated with temperature. At 40 ℃, 88.4% relative enzyme activity was retained,
440
while 62.7% relative enzyme activity was retained at 50 ℃, indicating that the 20
441
enzyme protein is stable in the temperature range of 30-50 ℃. However, only 27.1%
442
relative enzymatic activity was retained at 60 ℃, and the TS enzyme was completely
443
denatured at 70 ℃.
444
3.6. Phylogenetic analysis
445
Phylogenetic analysis of the amino acid sequence encoding TS in Camellia
446
gymnogyna Chang (Fig. S3) was compared with other plant-derived amino acids
447
encoding for N-methyltransferase, using MEGALIGN software. Results of the
448
analysis are shown in Fig. 3A. Based on the analyses of various tea tree species,
449
including Theobroma cacao, N-methyltransferase could be broadly classified into
450
three categories: those present in Camellia ptilophylla (BAE79732.1), Camellia
451
irrawadiensis (BAE79729.1), and Camellia japonica (BAG84612.1). Theobroma
452
cacao (BAE79730.1) and CgC are classified into the same category; Camellia sinensis
453
(ABP98983.1), Camellia crassicolumna (ALP01720.1) and Camellia taliensis
454
(ALP01721.1) belong to the same class; and Camellia kissii (BAG84615.1) and
455
Camellia petelotii (BAG84617.1) are part of the same class. It was shown that high
456
affinity exists between the amino acid sequence encoding TS in CgC and the
457
therobromine synthase of Camellia ptilophylla and Camellia irrawadiensis.
458
3.7. Leaf position-specific expression of TS transcript
459
qRT-PCR and western-blot analyses were performed on enzymes examined for
460
activity in two different leaf positions, the first leaf and fourth leaves, to demonstrate
461
differences in TS expression levels. The expression levels of the TS gene at mRNA
462
and protein levels were consistent, both being higher in the first leaf than those in the 21
463
fourth leaf (P < 0.05) (Fig. 3B and C), and both have a positive correlation with the
464
theobromine content.
465
3.8. Subcellular localization of TS
466
When the distribution of green fluorescence in cells is observed under a laser
467
confocal microscope, an excessively long dark culture time will result in no
468
observation of green fluorescence or a decrease in light intensity which influences the
469
observation. Because green fluorescent protein (GFP) is transiently expressed, it
470
cannot be maintained for extended periods of time. Experimental results are shown in
471
Fig. 3D, the red light corresponds to chloroplast self-luminescence, and green
472
fluorescence was a result of GFP luminescence, which in the overlay was observed in
473
the nucleus, indicating that TS is localized in the nucleus.
474
4. Discussion
475
Ordinarily, the dry weight content of caffeine in Camellia sinensis is 2~4%, relative
476
content of theobromine is approximately 0.05%, and that of theophylline is
477
approximately 0.002%; theacrine is predominantly distributed in the young bud and
478
leaves of Camellia assamica var. kucha, with a relative content of approximately
479
0.3~3%. Purine alkaloids have a variety of physiological effects, however there are
480
significant differences between individual compounds. Caffeine is the principal
481
constituent substance affecting the taste of tea. Appropriate caffeine content is
482
beneficial to human health, however, high contents give rise to central nervous system
483
(CNS) excitability, resulting in poor sleep or excessive excitement (Roehrs & Roth,
484
2008). Theacrine can improve monoamine neurotransmitter disorders and protect 22
485
neurons in animals, thus, it can act as an anti-depressant and improve memory (Xie et
486
al., 2009). Variations in plant purine alkaloid compositions are primarily affected by
487
species, environment, climate and processing methods. According to previous
488
research, Camellia sinensis contains abundant caffeine but lower levels of
489
theobromine and theophylline. Camellia ptilophylla contains chiefly theobromine and
490
lower levels of caffeine, which contributes to its ability to reduce blood pressure and
491
lessen CNS excitability (Wu et al., 2014). The dominant purine alkaloid in a new
492
camellia species, Camellia assamica var. kucha, is theacrine (Yang, Ye, Xu, & Jiang,
493
2007). CgC is a novel and scarce tea resource, with purine alkaloid composition and
494
proportions that differ significantly from those found in the classes of tea trees
495
reported above. Therefore, it is of great scientific significance to uncover the causes of
496
such purine alkaloid compositions in CgC and their characteristics, as it would offer a
497
deeper insight into classes of tea trees, and importantly, into the metabolic pathways
498
of purine alkaloids.
499
Q-Sepharose Fast Flow strong anion exchange chromatography, Sephadex G-75 gel
500
chromatography and ultrafiltration membranes were used to isolate and purify the
501
theobromine synthase monomer. The low recovery and reduced activity of the
502
enzyme was mainly attributed to limited raw materials, the choice of chromatographic
503
methods and the poor stability of N-methyltransferase in vitro. Hence, isolated
504
N-methyltransferase possessed lower activity, and the purified theobromine synthase
505
amount was sufficient for purification at electrophoresis level. Based on the protein
506
standards, the molecular weight of theobromine synthase was estimated to be 23
507
approximately 62 kDa. Therefore, determination of the amino acid sequence, accurate
508
molecular weight, functional structure relationships and enzymatic properties of
509
theobromine synthase requires further exploration.
510
It is known from the purine alkaloid synthesis pathway (Fig. S1) that caffeine
511
biosynthesis can utilize pseudo-xanthine and theophylline as precursors in the salvage
512
pathway, in addition to the primary pathway with theobromine. In the experiment
513
with various substrate and the enzyme monomer, it was found that purified caffeine
514
from CgC could not participate as a TS enzyme substrate. This is in contrast to results
515
obtained in a substrate specificity study of N-methyltransferase of Camellia assamica
516
var. kucha reported by Zheng (Zheng, Ye, Kato, Crozier, & Ashihara, 2002), who
517
found that paraxanthine, 7-methylxanthine, theobromine and caffeine were utilized as
518
methyl donors in the reaction with theacrine synthase. We concluded that there are
519
alternative enzymatic pathways involved in theacrine synthase in CgC, that are
520
distinct from the methylation site and catalytic action of the TS enzyme in Camellia
521
sinensis, Camellia ptilophylla and Camellia assamica var. kucha. This indicated that
522
there are significant differences in the recognition of key sites between theobromine
523
synthases purified from different plants and their methyl receptors, i.e., their
524
substrates. Unfortunately, since the Camellia assamica var. kucha synthetic base
525
enzyme pathways and genes require further exploration, there is no way to tell if a
526
salvage pathway for caffeine production exists in CgC; or, whether altering the
527
contents of theobromine and caffeine would achieve regulation of theacrine content.
528
These questions require further studies in more detail. 24
529
5. Conclusion
530
This study
has verified that the purine alkaloids of CgC from Dayao Mountain
531
consisted of theobromine, caffeine and theacrine, and that theobromine content was
532
the highest, followed by that of theacrine, and the lowest being caffeine. This purine
533
alkaloid composition and ratio is clearly distinguishable from Camellia sinensis,
534
Camellia ptilophylla and Camellia assamica var. kucha. The leaching tissue
535
homogenate method, ammonium sulfate precipitation, dialysis, strong anion exchange
536
chromatography, gel filtration chromatography, and ultrafiltration were used to
537
partially purify theobromine synthase (TS) from CgC. The purified enzyme study in
538
which various substrates were tested showed that enzyme affinity was as follows:
539
7-methylxanthine > xanthosine > xanthine > paraxanthine. The Km values were in the
540
range of 41-197 μmol/L, however theobromine, caffeine and theophylline did not
541
participate as substrates in the enzymatic reaction. Moreover, the optimal reaction
542
time of theobromine synthase was 6 h, the optimum pH was 8.0, and the optimum
543
reaction temperature was 35 ℃. After treatment at 60 ℃ for 20 min, relative enzyme
544
activity was only 27.1%, and the enzyme became thoroughly inactivated with an
545
increase in temperature beyond 60 ℃. Subcellular localization experiments showed
546
that the TS gene was localized in the nucleus. In the qRT-PCR and Western-blot
547
protein expression experiments of the TS enzyme, regardless of whether it was at the
548
gene level or protein level, the expression level in the first leaves of the tea was higher
549
than that in the fourth leaves, that is, the expression in young leaves was higher than
550
that of the older leaves. Consequently, theobromine contents, TS gene expression and 25
551
TS protein expression were positively correlated with each other. This study provides
552
a unique perspective on N-methyltransferases in tea plants and will hopefully renew
553
interests into their further exploration.
554
Acknowledgement
555
We thank Drs. Jinqiang Chen for critical review of this manuscript, and we would
556
like to thank Editage (www.editage.cn) for English language editing. This work was
557
supported by the Fu Jian Province “2011Collaborative Innovation Center” Chinese
558
Oolong Tea Industry Innovation Center Special Project (J2015-75); Guangdong Tea
559
Industry Research System (2019LM1117).
560
Conflict of interest
561 562 563 564
The authors notify that are no conflicts of interest. Ethical approval This study does not involve any human or animal testing. Supplementary data
565
Fig. S1. Purine alkaloid synthesis and metabolic pathways.
566
Fig. S2. Morphological characteristic of Camellia gymnogyna Chang from Dayao
567 568 569
Mountain: plant type and leaves. Fig. S3. Nucleotide and predicted amino acid sequences of theobromine synthase in a wild tea plant (Camellia gymnogyna Chang) from Dayao Mountain, China.
570 571
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29
Figure captions Fig. 1. Isolation, purification and detection of purified TS in Camellia gymnogyna Chang from Dayao Mountain. (A) Q-Sepharose Fast Flow ion exchange chromatography elution profile of the TS enzyme. (B) Sephadex G-75 gel filtration of Q-Sepharose Fast Flow ion exchange eluent TS activity combined fractionation curve. (C) SDS-PAGE of TS purification processes (Marker, protein standards; Lane 1, Crude enzyme extract from CgC; Lane 2, 50~80% (NH4)2SO4 saturated precipitation fractionation; Lane 3, Elution of TS enzyme activity peak of Q-Sepharose Fast Flow ion exchange chromatography; Lane 4, Sephadex G-75 gel filtration of Q-Sepharose Fast Flow ion exchange eluent TS activity combined fractionation). (D) Western blotting profile of various TS preparations with an anti-theobromine synthase polyclonal antibody. Fig. 2. Properties of purified theobromine synthase. Effect of reaction time (A), pH (B), temperature (C) and thermal stability (D) on the relative activity of TS in CgC from Dayao Mountain. Fig. 3. Characteristics of the theobromine synthase gene from Camellia gymnogyna Chang (Dayao Mountain). (A) The neighbor-joining phylogenetic tree of N-methyltransferase members from various plants. TS mRNA (B) and protein (C) relative expression levels in different leaf positions of CgC. * means significant differences (P < 0.05). (D) Subcellular localization of the TS fluorescent protein produced by vector transient in N.benthamiana leaves (Bars = 19.00 μm).
30
List of chemical compounds (No.: FOODCHEM-D-19-03917) 1 Caffeine (PubChem CID:2519) 2 Theobromine (PubChem CID:5429) 3 Theophylline (PubChem CID:2153) 4 Theacrine (PubChem CID:75324) 5 7-methylxanthine (PubChem CID:68374) 6 Paraxanthine (PubChem CID:5687) 7 Xanthine (PubChem CID:1188) 8 Xanthosine (PubChem CID:64959)
TABLES Table 1. The contents of purine alkaloids in Camellia gymnogyna Chang from Dayao Mountain. Leaf positions
Camellia
Theobromine (mg/g)
Theacrine (mg/g)
Caffeine (mg/g)
Bud
34.45±1.37b
5.82±0.04a
1.73±0.05c
The first leaf
39.72±1.43a
6.33±0.05d
2.02±0.06a
The second leaf
23.82±1.29d
6.86±0.09c
1.86±0.03b
The third leaf
13.46±0.84e
7.36±0.12b
1.46±0.04d
The fourth leaf
14.37±0.99f
9.10±0.11a
0.51±0.02e
A bud and two leaf
31.57±1.61c
6.36±0.07d
1.94±0.05b
gymnogyna Chang
Note: Different small letters in same column data indicate significant difference at the P< 0.05 level.
31
Table 2. Summary of isolation and purification of the TS in Camellia gymnogyna Chang from Dayao Mountain. Volume
Enzymatic activity
Total protein
Specific activity
Purification
Yiel
(mL)
(U)
(mg)
(U/mg)
fold
(%)
384
1463
154.34
9.48
1.00
100
28
837
66.59
12.57
1.32
57.2
Q-Sepharose F F
14
91
1.20
75.83
8.00
6.22
Sephadex G-75
6
29
0.16
181.25
19.12
1.98
1.5
17
0.05
340.00
35.87
1.16
Step Crude enzyme 50~80% (NH4)2SO4 precipitate
Ultrafiltration
Table 3. Kinetic properties of TS in Camellia gymnogyna Chang from Dayao Mountain. NO.
Substrates
1
7-methylxanthine
Chemical formula
Km (μmol/L)
Vmax (U/mg)
Kcat (1/S)
41
237
301
2
Paraxanthine
197
65
82
3
Xanthine
144
89
112
32
4
Xanthosine
86
192
237
5
Theobromine
nd
nd
nd
6
Caffenine
nd
nd
nd
7
Theophylline
nd
nd
nd
nd, Not detected.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Highlights
Camellia gymnogyna Chang tea plant, found in Guangxi Province contains three different purine alkaloids. 33
Theobromine synthase was from Camellia gymnogyna Chang, purified, and characterized.
7-methylxanthine was a favorable substrate for theobromine synthase.
Full length cloning, subcellular localization, and polygenetic tree analysis of the theobromine synthase gene.
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