Reductase-catalyzed tetrahydrobiopterin regeneration alleviates the anti-competitive inhibition of tyrosine hydroxylation by 7,8-dihydrobiopterin

Article abstract of DOI:10.1039/d0cy01958e

l-Tyrosine hydroxylation by tyrosine hydroxylase is a significant reaction for preparing many nutraceutical and pharmaceutical chemicals. Two major challenges in constructing these pathways in bacteria are the improvement of hydroxylase catalytic efficiency and the production of cofactor tetrahydrobiopterin (BH4). In this study, we analyzed the evolutionary relationships and conserved protein sequences between tyrosine hydroxylases from different species by PhyML and MAFFT. Finally, we selected 7 tyrosine hydroxylases and 6 sepiapterin reductases. Subsequently, the function of different groups was identified by a combined whole-cell catalyst, and a series of novel tyrosine hydroxylase/sepiapterin reductase (TH/SPR) synthesis systems were screened including tyrosine hydroxylase (from Streptosporangium roseum DSM 43021 and Thermomonospora curvata DSM 43183) and sepiapterin reductase (from Photobacterium damselae, Chlorobaculum thiosulfatiphilum and Xenorhabdus poinarii), namely as SrTH/PdSPR, SrTH/CtSPR, SrTH/XpSPR and TcTH/PdSPR, which can synthesize l-Dopa by hydroxylating l-tyrosine in Bacillus licheniformis. Furthermore, we analyzed the characterization of SrTH by enzyme catalysis and demonstrated that 7,8-dihydrobiopterin (BH2) formed by BH4 autooxidation was an anticompetitive inhibitor on SrTH. Finally, pure dihydropteridine reductase from Escherichia coli (EcDHPR) was added to the solution, and l-Dopa could be continually synthesized after 3 h, which was improved by 86% at 6 h in the catalytic reaction by SrTH. This indicates that BH4 regeneration can alleviate the inhibition by BH2 during tyrosine hydroxylation. This study provides a good starting point and theoretical foundation for further modification to improve the catalytic efficiency of tyrosine hydroxylation by tyrosine hydroxylase.

Full text of DOI:10.1039/d0cy01958e

Catalysis
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Technology
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Reductase-catalyzed tetrahydrobiopterin
regeneration alleviates the anti-competitive
inhibition of tyrosine hydroxylation by
Cite this: Catal. Sci. Technol., 2021,
11, 3128
7,8-dihydrobiopterin†
abc
abc
abc
abc
Yinbiao Xu,
Youran Li,*
Leyun Li,
and Guiyang Shi
Liang Zhang,
abc
abc
Zhongyang Ding
*
L-Tyrosine hydroxylation by tyrosine hydroxylase is a significant reaction for preparing many nutraceutical
and pharmaceutical chemicals. Two major challenges in constructing these pathways in bacteria are the
improvement of hydroxylase catalytic efficiency and the production of cofactor tetrahydrobiopterin (BH4).
In this study, we analyzed the evolutionary relationships and conserved protein sequences between
tyrosine hydroxylases from different species by PhyML and MAFFT. Finally, we selected 7 tyrosine
hydroxylases and 6 sepiapterin reductases. Subsequently, the function of different groups was identified by
a combined whole-cell catalyst, and a series of novel tyrosine hydroxylase/sepiapterin reductase (TH/SPR)
synthesis systems were screened including tyrosine hydroxylase (from Streptosporangium roseum DSM
4
3021 and Thermomonospora curvata DSM 43183) and sepiapterin reductase (from Photobacterium
damselae, Chlorobaculum thiosulfatiphilum and Xenorhabdus poinarii), namely as SrTH/PdSPR, SrTH/
CtSPR, SrTH/XpSPR and TcTH/PdSPR, which can synthesize L-Dopa by hydroxylating L-tyrosine in Bacillus
licheniformis. Furthermore, we analyzed the characterization of SrTH by enzyme catalysis and
demonstrated that 7,8-dihydrobiopterin (BH2) formed by BH4 autooxidation was an anticompetitive
inhibitor on SrTH. Finally, pure dihydropteridine reductase from Escherichia coli (EcDHPR) was added to
the solution, and L-Dopa could be continually synthesized after 3 h, which was improved by 86% at 6 h in
the catalytic reaction by SrTH. This indicates that BH4 regeneration can alleviate the inhibition by BH2
during tyrosine hydroxylation. This study provides a good starting point and theoretical foundation for
further modification to improve the catalytic efficiency of tyrosine hydroxylation by tyrosine hydroxylase.
Received 7th October 2020,
Accepted 15th March 2021
DOI: 10.1039/d0cy01958e
rsc.li/catalysis
pharmaceutical chemicals, such as melanin-like biomaterials,
Introduction
4
–8
plant alkaloids, betalain pigments and monolignols.
In
L-Tyrosine hydroxylation is the rate-limiting step in the central
nervous system and adrenal gland, where its role is to catalyze
the first step in the biosynthesis of neuroactive chemicals,
including L-Dopa, the catecholamines dopamine (DA),
animals, tyrosine hydroxylation is achieved by tyrosinase and
tyrosine hydroxylase requiring tetrahydrobiopterin (BH4) as a
2
,3
native cofactor.
Microbial L-tyrosine hydroxylation is
generally achieved by tyrosinase and 4-hydroxyphenylacetate-
1
,2
11,13–18
noradrenaline (NA) and adrenaline in animals.
3-hydrolase (HpaBC).
Simultaneously, L-tyrosine hydroxylation also plays an
important role in preparing multiple nutraceutical and
Tyrosinase is a widely distributed multicopper oxygenase in
bacteria, fungi, plants, and animals that has bifunctional
15–17
activity on monophenols and o-diphenols.
It is an internal
monooxygenase requiring molecular O and two electrons from
substrate in the catalysis process. The oxidase reaction from
2
a
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of
Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, P. R. China
b
o-diphenol to o-quinone is much more rapid than the
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan
1
7
University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, P. R. China.
E-mail: liyouran@jiangnan.edu.cn, gyshi@jiangnan.edu.cn
oxygenation reaction from monophenol to o-diphenol. L-Dopa
can be oxidized to L-dopaquinone via the diphenolase (DP)
c
Jiangsu Provisional Research Center for Bioactive Product Processing Technology,
19
activity of tyrosinase, eventually turning into melanin. HpaBC
Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, P. R.
China
is a native enzyme complex in E. coli that is able to hydroxylate
multiple substrates, such as 4-hydroxyphenylacetic acid,
tyrosine, p-coumaric acid and 4-tyrosol, with NADH as a native
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cy01958e
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cofactor for hpaC reducing FAD to FADH2 which is used for Experimental section
9
–14
substrate hydroxylation by hpaB.
HpaBC was used to
Computer-assisted sequence analysis
1
1,13,14
synthesize L-Dopa from glucose in previous study.
The protein sequences in this study were obtained from the
NCBI GenBank database (http://www.ncbi.nlm.nih.gov).
Multiple sequence alignments of protein sequences were
performed with the program MAFFT (MAFFT-linsi) v7.471
(ref. 26) and visualized with ESPript 3.0 (ref. 27) to identify
strictly conserved amino acid residues. The maximum-
likelihood phylogenetic tree of tyrosine hydroxylase was
created by PhyML v3.0 after selecting the best amino acid
However, a large amount of melanin would be formed and a
lot of tyrosine remained in the fermentation broth, which
causes loss of carbon flux to L-Dopa and its downstream high-
value products, such as hydroxytyrosol, plant alkaloids,
4–8,25
betalain pigments and monolignols.
It is worth noting that
tyrosine hydroxylase does not have diphenolase (DP) activity. In
the previous study, Jay D. Keasling and Taek Soon Lee used TH
from mouse for the production of L-Dopa and hydroxytyrosol
2
8
25
substitution model by ProtTest v3.4.2.
with MH4 as cofactor in E. coli. The conserved amino acid
positions and substrate binding site of tyrosine hydroxylase
2
1,35,39
Microorganisms and cultivation conditions
from animals have been elaborated.
There is no report
on tyrosine hydroxylase from microorganisms. The specific
enzyme activity of tyrosine hydroxylase from human and mouse
B. licheniformis CICIM B6902, stored in the Culture and
Information Center of Industrial Microorganisms of China
Universities (CICIM-CU), was used in this study, and the
pMA5 plasmid was provided by Beinuo Life Science
(Shanghai, China). E. coli DH5α (CICIM B0006) and B.
licheniformis CICIM B6902 seed cultures were maintained in
150 mL flasks containing 15 mL of lysogeny broth (LB) at 37
°C and 200 rpm on a rotary shaker for 12 h. The following
antibiotics were added as appropriate to the growth media:
−1
−1
is 1017 nmol min (mg of protein) at 25 °C and 2.78 nmol
−
1
−1
min (mg of protein) at 30 °C in 10 mM NaHepes buffer (pH
7
2
0,40
.0).
However, the specific enzyme activity of TH from
animals at 37 °C reduced to 15.4% of that at 30 °C, which
indicates that tyrosine hydroxylase from animals is sensitive to
4
0
high temperature. Additionally, tyrosine hydroxylase from
animals can be inhibited by many chemicals, such as L-tyrosine
−
1
−1
(
5
over 100 μM), oxygen (higher than 4.8%), 4-amino-BH4,
-methyl-BH4 and 8-M-6,7-DMPH4.
30 μg mL kanamycin and 100 μg mL ampicillin. For
enzyme expression, cells were grown at 37 °C and 250 rpm in
fermentation medium (pH 7.0) containing (per liter) lactose
(30 g), yeast extract (24 g), peptone (12 g), K HPO ·3H O
2
0,21
The hydroxylation of L-tyrosine by tyrosine hydroxylase
requires the participation of the cofactor BH4, L-tyrosine and
oxygen. Fortunately, BH4 can be successfully synthesized in
2
4
2
(16.43 g) and KH PO (2.3 g).
2
4
−
1
E. coli, reaching 718 mg
L
by AG14/(pSTV28GPS,
pMW218guaBA) by using GCHI from B. subtilis which is also
Construction of plasmids and recombinant strains
present in B. licheniformis and has lower K value than FolE
m
2
2,47,48
All the constructed strains and plasmids are listed in Table
S1,† and all the primers for constructing plasmids are listed
in Table S2.† The codon-optimized genes from different
microorganisms were synthesized by Synbio Technologies
(Suzhou, China). All synthesized genes were submitted to the
NCBI GenBank database. The GenBank accession numbers
and sequences of all genes are listed in Table S3.† The PCR
products were digested with NdeI and BamHI and cloned into
NdeI–BamHI-digested pMA5 after agarose gel purification.
The ligation mixtures were first transformed into chemically
competent E. coli DH5α cells. The positive recombinants were
first selected by the phenotype showing ampicillin resistance
and further confirmed by PCR.
from E. coli.
The titer of 5-hydroxytryptophan, also
−
1
requiring BH4 as a cofactor, can reach up to 1.61 g L in
shaking flasks by E. coli, indicating that it is feasible to
efficiently synthesize hydroxyl chemicals with BH4 as a
3
,23,24
cofactor.
However, this goal by tyrosine hydroxylase has
not been achieved, although a new pathway producing
L-DOPA by tyrosine hydroxylase has been demonstrated in the
progress of synthesizing hydroxytyrosol in E. coli, which may
be due to the poor catalytic efficiency at 37 °C of tyrosine
2
5,40
hydroxylase from mouse.
Therefore, we tried to screen
tyrosine hydroxylase/sepiapterin reductase (TH/SPR) synthesis
systems from microorganisms.
In this study, we describe tyrosine hydroxylation by tyrosine
hydroxylase from Streptosporangium roseum DSM 43021 (SrTH)
in B. licheniformis and the characterization of SrTH during the
catalytic process; that is, (1) screening of tyrosine hydroxylases
and sepiapterin reductases from NCBI by computer-assisted
sequence analysis, (2) functional identification of TH/SPR dual-
enzyme synthesis systems by combined whole-cell catalyst, (3)
inhibition analysis of dihydrobiopterin during crude enzyme
reaction, (4) characterization of inhibition by BH2 on SrTH
partially purified via ion exchange chromatography and (5)
evaluating the effectiveness of pure dihydropteridine reductase
from E. coli (EcDHPR) addition on the reaction catalyzed by
crude SrTH.
Enzyme assays
The TH activity was assayed at 37 °C, using an incubation
mixture containing 20 mM sodium phosphate buffer (pH 7.0),
−1
−1
50 mg L L-tyrosine, 0.25 mmol L BH4 and 0.25 μM Fe SO .
2
4
The enzyme was preincubated for 10 min with 0.25 μM Fe SO
and 0.25 mmol L BH4 before the reaction was started by
2
4
−1
addition of L-tyrosine. The reaction kept 3 min at 37 °C and 2
−1
mmol L sodium periodate was added into the solution. The
mix kept at 25 °C in the dark for 10 min. All experiments were
done at a final volume of 222 μL and 200 μL was taken into a
50
96-well plate for measurement at 475 nm.
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Hydroxylation of tyrosine by synergetic whole-cell catalysts
Analysis of the interaction between SrTH and pterins by the
Autodock 4.0 program
After verification by DNA sequencing, the plasmids harboring
different genes were transferred by electroporation into B.
licheniformis. The positive recombinants were first selected by
the phenotype showing kanamycin resistance and further
confirmed by PCR. B. licheniformis transformants were
inoculated into 50 mL of fermentation medium supplied with
The sequence identity of complete sequence and protein
functional area is 22.29% and 38.02% between SrTH and
HuTH (PDB ID: 2xsn), which analyzed by searching for
3
1
templates in SWISS-MODEL. The SrTH model was built by
3
1
SWISS-MODEL with HuTH as template and structurally
evaluated by the SAVES program (https://servicesn.mbi.ucla.
edu/SAVES/) (Fig. S1†), which runs PROCHECK, Verify3D,
−1
3
0 μg mL kanamycin and 3% (v/v) seed cultures in 250 mL
flasks and cultured at 37 °C and 250 rpm for 24 h. After
fermentation, the OD600 was measured. The cells were collected
by centrifugation at 4 °C and 12000 rpm for 10 min, washed
3
2
What_Check, and ERRAT at the same time. The PDB
structures of BH4 and dihydrobiopterin were retrieved from
PubChem Compound of the NCBI GenBank database.
Protein–ligand interaction studies between SrTH and pterins
were carried out using Discovery Studio 3.0 (Accelrys) and
Autodock 4.0 programs.
−1
twice with 10 mmol L sodium phosphate buffer (pH 7.0) and
resuspended in the above buffer to an OD600 of 20. The two
kinds of cells expressing TH or SPR were mixed 1 : 1 in a two-
−1
by-two combination. In the process, 2 g L L-tyrosine was
added, and the reaction was at 37 °C and 250 rpm for 24–36 h
with 30 mL in 250 mL flasks. After combining whole-cell
catalysts, the samples were centrifuged at 4 °C and 12000 rpm
for 20 min, and the supernatant was treated with 5%
trichloroacetic acid. After 12 h at 4 °C, the samples were
centrifuged for 20 min at 4 °C and 12000 × g, and a total
volume of 50 μL of supernatant was added into an HPLC vial.
Product inhibition analysis of SrTH by dihydrobiopterin
−1
−1
In this study, we supplemented 1.5 g L tyrosine, 5 g L
−1
2+
−1
ascorbic acid, 100 μmol L Fe , 0.25 mmol L BH4 per
−1
hour and 1 g L crude enzyme powder containing SrTH per
hour in the enzymatic process. During the progress of
L-tyrosine hydroxylation by SrTH, we supplemented with 0.64
−
1
−1
mmol
L
6,7-dihydrobiopterin or
1
mmol
L
L-Tyrosine hydroxylation by SrTH in vitro
7,8-dihydrobiopterin in the solution for the characterization
of inhibition of dihydrobiopterin on hydroxylase. After
catalyst at 37 °C 180 rpm for 3 h, L-Dopa in the supernatant
was detected by HPLC.
After verification by DNA sequencing, the plasmids harboring
SrTH were transferred by electroporation into B. licheniformis.
The positive recombinants were first selected by the
phenotype showing kanamycin resistance and further
confirmed by PCR. B. licheniformis transformants were
inoculated into 50 mL of fermentation medium supplied with
Protein purification via DEAE-cellulose ion exchange
chromatography
−
1
3
0 μg mL kanamycin and 3% (v/v) seed cultures in 250 mL
The mobile phase was filtered through a 0.22 μm-pore-size
membrane and degassed for 20 min before use. A DEAE-
cellulose column (column volume 5 mL) was pre-equilibrated
flasks and cultured at 37 °C and 250 rpm for 24 h. After
fermentation, the OD600 was measured. The cells were
collected by centrifugation at 4 °C and 12 000 rpm for 10
4
9
with 20 mM sodium phosphate buffer (pH 7.0) for 20 min.
The protein sample was linearly eluted with sodium
−
1
min, washed twice with 10 mmol L sodium phosphate
buffer (pH 7.0), and resuspended in the above buffer to an
−
1
phosphate buffer containing NaCl from 0–1 mol L at a flow
−
1
−
1
OD600 of 5. After treatment with 5 g L lysate in a 37 °C
incubator for 30 min, the cell suspension was ultrasonically
broken and centrifuged at 4 °C and 12 000 rpm for 20 min to
obtain the crude enzyme solution. The crude enzyme solution
was frozen at −70 °C for 12 h and lyophilized into powder
using a Benchtop Pro freeze-dryer (SP Scientific, New York,
USA). The protein content was quantified by the Bradford
rate of 2 ml min . Fractions were collected every 2 min. The
absorbance of these fractions was measured at 280 nm. The
2
9
protein content was quantified by the Bradford assay using
bovine serum albumin (BSA) as a standard.
Fluorescence measurements of pterin-SrTH systems
−
1
29
The protein concentration was 44 mg L . The concentration
assay using bovine serum albumin (BSA) as a standard. The
gene product of SrTH was confirmed by SDS polyacrylamide
gel electrophoresis (SDS-PAGE) using a 12% polyacrylamide
−
1
of BH4/BH2/BH2 with adding 10 μmol L BH4/PH2 were
−
1
prepared from 0 to 20 μmol L for SrTH. The fluorescence
scanning was achieved in the range of 300–450 nm with 265
nm of excitation wavelength at four temperatures (298, 303,
3
0
gel as described by U. K. Laemmli. Protein bands were
stained using a protein stains Q kit (Sangon Biotech,
Shanghai, China).
4
5
310 and 315 K) according to the previous study.
−
1
In the reaction, we supplemented with 1.7 g L tyrosine, 5
−
1
−1
2+
Characterization of inhibition by BH2 on SrTH
g L ascorbic acid and 100 μmol L Fe one time. At the
−
1
same time, 1 g L crude enzyme powder containing SrTH
Kinetic studies on the interaction between tyrosine
hydroxylase and L-tyrosine, oxygen and tetrahydropteridines
−
1
and 0.25 mmol L BH4 were supplemented in the solution
per 1 hour. After catalyst at 37 °C 180 rpm for 6 h, L-Dopa in
the supernatant was detected by HPLC.
2
0,21,33
were previously performed.
In this study, we
−
1
−1
supplemented 1.5 g L tyrosine, 5 g L ascorbic acid, 100
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−
1
2+
−1
μmol L Fe and 20 mg L SrTH purified via ion exchange
mollusks, arthropods, green plants, fungi and chordates. The
sequences were used to construct a maximum-likelihood
phylogenetic tree by the PhyML program. Finally, we
screened out 7 tyrosine hydroxylases from Actinobacteria with
similar conservative amino acid positions as shown in Fig. 1.
The accession numbers for these sequences are shown in the
figure. Analysis of amino acid sequences indicated that
tyrosine hydroxylase from Actinobacteria is the earliest in
evolutionary time and has the farthest relationship with
chordates (Fig. 1A).
chromatography in the enzymatic process. At the same time,
.05–0.25 mmol
,8-dihydrobiopterin were supplemented in the enzyme
reaction. After 1 h at 37 °C, the reaction was terminated by a
final concentration of 5% trichloroacetic acid, and the
production of L-Dopa was detected by HPLC. The kinetic
parameters Km and Vmax were analyzed by a Lineweaver–Burk
plot (double reciprocal plot), which utilizes 1/[S] as the X axis
and 1/V as the Y axis (1).
−
1
−1
0
L
BH4 and 0–0.4 mmol
L
7
1
v
Km
1
1
To analyze the conserved amino acid positions of tyrosine
hydroxylase from different species during the evolution
process, multiple sequence alignment (MSA) with secondary
structure depiction (HuTH as template) was performed on
these sequences through MAFFT and ESPript (Fig. 1B). We
found that the consensus of 20 amino acid positions was
¼
·
þ
(1)
Vmax ½Sꢀ Vmax
i
K value was analyzed by a Lineweaver–Burk plot (double
reciprocal plot), which utilizes 1/[S] as the X axis and 1/V as
the Y axis (2).
ꢀ
ꢁ
100% in all species, including Pro270, Gly284, Phe285,
1
v
Km
1
1
½Iꢀ
Ki
¼
·
þ
1 þ
(2)
Phe299, Phe308, Arg315, Pro320, Pro326, Asp327, His330,
Gly334, His335, Gly352, Trp371, Glu375, Gly377, Gly389,
Ala390, Ser394, and Glu398. Notably, some important amino
acid positions were not absolutely conserved, such as Gly292,
Leu293, Leu294 and Tyr370, which are important for pterin
binding. For example, tyrosine hydroxylase (PDB ID:
Vmax ½Sꢀ Vmax
where V is the rate of the reaction, Vmax is the maximum rate
of reaction, K is the Michaelis–Menten constant and
indicates the affinity of the substrate to the enzyme, K is the
inhibition constant of inhibitor, [S] is the substrate
concentration and [I] is the inhibitor concentration.
m
i
EFE75625.1) had
a G292A amino acid substitution in
Streptomyces filamentosus NRRL15998, which may damage
flexibility in this loop region and affect pterin binding.
Similarly, the tyrosine hydroxylase (PDB ID: KPI05405.1) from
Actinobacteria bacterium OK006 revealed L293T amino acid
substitutions, which may influence the formation of
Analysis of L-Dopa, BH4 and dihydrobiopterin
The samples were treated with a final concentration of 5%
trichloroacetic acid. After incubation for 12 h at 4 °C, the
samples were centrifuged for 20 min at 4 °C and 12 000 ×
3
5
hydrogen bonds from C-1′OH of pterin.
The tyrosine
g, and
a total volume of 50 μL of supernatant was
hydroxylases (PDB ID: KPI20637.1, EGD43416.1, ANZ14715.1,
AJC54316.1 and ACY06282.1) also revealed L293I, L293I,
L293V, L293V and L293V amino acid substitutions in
Actinobacteria bacterium OV450, Nocardioidaceae bacterium
Broad-1, Streptomyces noursei ATCC 11455, Streptomyces sp.
769 and Streptomyces flaveolus, respectively.
transferred to an HPLC vial. The supernatant was analyzed
by a Thermo HPLC UltiMate 3000 comprising a quaternary
pump, an autosampler, and a UV detector. Samples were
separated on a Waters XSelect®HSS T3 C18 column. The
mobile phase for L-Dopa analysis consisted of 97% 10
−
1
mmol L
sodium acetate (HPLC) and 3% acetonitrile
In this study, we chose 7 tyrosine hydroxylases, namely,
(
HPLC). The mobile phase for BH4 and dihydrobiopterin
−
1
KPI05405.1,
ACQ81820.1,
ABW14799.1,
CCH30170.1,
detection consisted of 97% 30 mmol L sodium acetate
3
4
ACZ90985.1, ADG87750.1 and ADG87750.1, which were from
Actinobacteria bacterium OK006, Beutenbergia cavernae DSM
(HPLC) and 3% methanol (HPLC). The mobile phase was
filtered through 0.22 μm-pore-size membrane and
a
1
4
2333, Frankia sp. EAN1pec, Saccharothrix espanaensis DSM
4229, Streptosporangium roseum DSM 43021, Thermobispora
degassed for 20 min before use. The column temperature
was maintained at 40 °C, while detection was monitored
at wavelengths of 280 nm for L-Dopa and 246 nm for BH4
and dihydrobiopterin. The injection volume of the samples
bispora DSM 43833 and Thermomonospora curvata DSM
3183, respectively, for further research. The synthesized
4
−
1
sequences and GenBank accession numbers of 7 tyrosine
hydroxylases are listed in Table S3.†
was set at 10 μL with a flow rate of 1 mL min . A
standard curve was constructed from serial dilutions of a
standard stock solution.
Functional identification of TH/SPR dual-enzyme synthesis
systems
Results
Computer-assisted sequence analysis of tyrosine hydroxylases
Preliminary studies have shown that it requires the
participation of BH4, a cofactor produced by the folate
synthesis pathway, in the process of catalyzing L-Dopa
The conserved amino acid positions and substrate binding
site of tyrosine hydroxylase from animals have been
2
1,35,39
25
elaborated.
Forty-one sequences of TH from different
synthesis by TH. To synthesize BH4 in B. licheniformis, we
species were obtained from the NCBI GenBank database,
which belonged to Actinobacteria, flatworms, nematodes,
screened 6 sepiapterin reductases from microorganisms
according to the tyrosine hydroxylase screening method. The
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Fig. 1 Computer-assisted sequence analysis of tyrosine hydroxylases. A maximum-likelihood phylogenetic tree using amino acid sequences built
using the PhyML software after selecting the best amino acid substitution model by ProtTest v3.4.2; B multiple sequence alignments of protein
sequences harboring secondary structure depiction (HuTH as template) performed with the program MAFFT (MAFFT-linsi) v7.471 and visualized
with ESPript 3.0. (a) amino acid positions of HuTH from 266 to 306; (b) amino acid positions of HuTH from 307 to 402.
−
1
synthesized sequences and GenBank accession numbers of 6
sepiapterin reductases are listed in Table S3.†
rpm for 36 h (Fig. 2). During the catalytic process, 2 g L
L-tyrosine was added into the reaction solution as the substrate
for L-Dopa. Obviously, the color of some combination solutions
changed to black after whole cell transformation, indicating
that L-Dopa was successfully synthesized in the catalytic
process. L-Dopa will be oxidized to dopaquinone in the air and
eventually become melanin. We detected L-Dopa of the 42
In order to quickly and efficiently screen out functional TH/
SPR dual-enzyme synthesis systems, we use combined whole
cell to catalyse L-tyrosine for producing L-Dopa. The resting
cells of tyrosine hydroxylases were combined pairwise with that
of sepiapterin reductases for L-Dopa production at 37 °C 250
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Hydroxylation of L-tyrosine with external BH4 supplementation
BH4 is the limiting factor in the process of tyrosine
2
4
hydroxylase catalysis. In this study, we attempted to use
SrTH to catalyze tyrosine for the synthesis of L-Dopa without
external BH4 supplementation. However, L-Dopa cannot be
detected, indicating that SrTH is also a kind of BH4-
dependent tyrosine hydroxylase.
To identify other bottlenecks in the enzymatic process, we
retained sufficient enzyme, L-tyrosine, ascorbic acid (Fig. S2†)
and BH4 levels in the crude enzyme reaction. To ensure that
SrTH can be continuously added, the resulting enzyme
solution after cell disruption was centrifuged and lyophilized
to obtain a powder. The enzyme powder was dissolved in
ddH O and analyzed using SDS-PAGE. Fig. 3A shows that
2
SrTH had been successfully expressed in B. licheniformis. In
the enzymatic process, we supplemented the reaction with
−
1
−1
−1
2+
1
.7 g L tyrosine, 5 g L ascorbic acid and 100 μmol L Fe
−
1
one time. At the same time, 1 g L crude enzyme powder
containing SrTH and 0.25 mmol L BH4 was supplemented
per hour. L-Dopa was continuously synthesized in the first
three hours, and its content increased to 90 mg L at 3 h.
However, L-Dopa did not continue to accumulate after 3 h
−
1
−
1
(
Fig. 3B). At the same time, we prepared the crude enzyme
powder of other 6 screened enzymes (TcTH, TbTH, FsTH,
BcTH, AbTH and SeTH) according to the same method as
before. Due to low catalytic efficiency of TbTH, FsTH, BcTH,
AbTH and SeTH as shown in Fig. 2 and S17,† L-Dopa
synthesized in vitro by crude enzyme powder could not reach
the limit of detection of HPLC. Only TcTH can synthesize
37.39 mg L L-Dopa in the catalysis process as shown in Fig.
S3,† which was much lower than that by SrTH. We analyzed
the substances present in the reaction found that when BH4
was continuously added, dihydrobiopterin accumulated
continuously, which may be the reason why L-Dopa did not
accumulate continuously (Fig. S4†).
Fig.
2 Functional identification of TH/SPR dual-enzyme synthesis
−
1
systems by combined whole-cell catalyst. The two kinds of cells
expressing TH or SPR were mixed by OD600 10 : OD600 10 in two-by-
two combination with 10 mmol L sodium phosphate buffer (pH 7.0).
−1
−
1
In the process, 2 g L L-tyrosine was added and the reaction was at 37
C, 250 rpm for 24–36 h with 30 mL in the 250 mL flasks. After
°
combined whole-cell catalyst by different groups, the color change
was recorded and L-Dopa in the supernatant was detected by HPLC.
All assays were performed in triplicate.
Subsequently, Autodock 4.0 was used to research the
protein–ligand interactions between SrTH and BH4,
6
,7-dihydrobiopterin or 7,8-dihydrobiopterin. We found that
combination solutions by HPLC. The synthesis of L-Dopa was
completely undetectable in 7 of 42 combinations, and the
L-Dopa concentration of 18 combinations was less than 5 mg
the other two substances interacted with the active center of
binding BH4 in SrTH. Glu163 of SrTH can simultaneously
form hydrogen bonds with BH4, 6,7-dihydrobiopterin and
−1
L . We found that the combinations with high L-Dopa content
basically focused on the combinations containing SrTH and
TcTH. There were four combinations with L-Dopa
7
7
,8-dihydrobiopterin (Fig. 4). In particular, BH4 and
,8-dihydrobiopterin have four common amino acid sites for
interaction, namely, Leu125, Phe131, His167 and Glu163
Fig. 4C). The docking results further indicate that the
−1
concentrations above 50 mg L , including TcTH/PdSPR (68.59
(
−1
−1
mg L ), SrTH/PdSPR (66.24 mg L ), SrTH/CtSPR (60.11 mg
accumulation of BH4 oxidation products is likely to inhibit
the synthesis of L-Dopa in the enzyme reaction.
−1
−1
L ), and SrTH/XpSPR (53.87 mg L ). According to the
phylogenetic tree (Fig. 1A), SrTH displayed a close relationship
to TcTH. Both of these proteins play a potential role in the
production of L-Dopa. Although the results were novel for
producing L-Dopa by TH, the production of L-Dopa by
combined whole-cell catalyst was lower than that of the other
reported L-Dopa synthesis systems, such as HpaBC and
tyrosinase. To unravel other limiting factors in the enzyme
reaction, we characterized the properties of L-tyrosine
hydroxylation by TH in the enzymatic process.
Inhibition of enzyme reaction by dihydrobiopterin
BH4 is extremely unstable and will be oxidized to
6,7-dihydrobiopterin, which can be naturally converted to
7,8-dihydrobiopterin (Scheme 1).
of BH4 at room temperature was analyzed by HPLC. We
found that the oxidation product of BH4 was a mixture of
6,7-dihydrobiopterin and 7,8-dihydrobiopterin during the
3
4,36
The oxidation process
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Fig. 3 Hydroxylation of L-tyrosine with external BH4 supplementation. A SDS-PAGE analysis of crude enzyme powder containing SrTH. Protein
content was quantified by the Bradford assay using bovine serum albumin (BSA) as standard. 10 μg protein of (M) molecular weight marker, (lane 1)
Bacillus licheniformis (blank) and (lane 2) Bacillus licheniformis (SrTH) was analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using
1
2% polyacrylamide gel. Protein bands were stained by silver staining using a Protein Stains Q kit; B production of L-Dopa by crude enzyme powder
−
1
−1
−1
2+
containing SrTH. In the reaction, we supplemented with 1.7 g L tyrosine ( ), 5 g L ascorbic acid and 100 μmol L Fe at a time. At the same
−
1
−1
time, 1 g L crude enzyme powder containing SrTH and 0.25 mmol L BH4 ( ) were supplemented in the solution per 1 hour. After catalyst at
37 °C 180 rpm for 6 h, L-Dopa (■) in the supernatant was detected by HPLC. The dash lines indicate the feed points.
reaction and was finally 7,8-dihydrobiopterin at the end of
the process. It was difficult to obtain high-purity
,7-dihydrobiopterin during the oxidation of BH4 (Fig. S5
6
and S6†). This goal was still not achieved when increasing
the temperature and the dissolved oxygen content during
−
1
oxidation, and only 0.32 mmol L 6,7-dihydrobiopterin could
be obtained after incubation at 37 °C 250 rpm for 24 h
(
(
Fig. 5A). BH4 was converted to 7,8-dihydrobiopterin at 60 h
Fig. S7†). To study the influence of 6,7-dihydrobiopterin on
the enzyme reaction, we tried to prepare high-purity
6
2
,7-dihydrobiopterin
,6-Dichloroindophenol is a redox agent that can be used to
using
redox
agents.
detect ascorbic acid and quickly oxidize BH4 to
3
7
6
2
2
,7-dihydrobiopterin. In this study, equimolar BH4 and
,6-dichloroindophenol were mixed uniformly at 37 °C and
50 rpm. The oxidation products were detected by HPLC. We
found that 6,7-dihydrobiopterin could accumulate to 0.64
−
1
mmol L at 1.3 h, a higher concentration than that obtained
without 2,6-dichloroindophenol (Fig. 5B and S8†).
−
1
Subsequently, 1 mmol L 7,8-dihydrobiopterin and 0.64
−
1
mmol L 6,7-dihydrobiopterin were added to the solution
during the crude enzyme reaction, and the synthesis of
L-Dopa was analyzed by HPLC (Fig. 6). We found that the
Fig. 4 Analysis of interaction between SrTH and pterins. The protein–
ligand interactions between SrTH and BH4, 6,7-dihydrobiopterin or
7
,8-dihydrobiopterin were studied using Discovery Studio 3.0 and
Autodock 4.0. A Tetrahydrobiopterin (BH4); B 6,7-dihydrobiopterin (q-
BH2); C: 7,8-dihydrobiopterin (BH2).
Scheme 1 The reaction path of BH4 auto-oxidation.
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−1
Fig. 5 Analysis of BH4 oxidation. A BH4 oxidation at natural condition. 1 mmol L BH4 was shaked at 37 °C 250 rpm and the chemicals BH4 (■),
BH2 ( ), q-BH2 ( ) were detected by HPLC. The dash line indicates the point for preparing 7,8-dihydrobiopterin (BH2); B BH4 oxidation by
−
1
2
,6-dichloroindophenol. The solution containing 1 mmol L BH4 and 2,6-dichloroindophenol was shaked at 37 °C 250 rpm and the chemicals
BH4 (■), BH2 ( ), q-BH2 ( ) were detected by HPLC the dash line indicates the point for preparing 6,7-dihydrobiopterin (q-BH2).
synthesis of L-Dopa was not affected by the addition of 0.64
It was previously reported that hTH1 would be
−
1
mmol L 6,7-dihydrobiopterin (Fig. 6A). It is worth noting
competitively inhibited by some BH4 analogs in the reaction,
such as 4-amino-BH4 and 5-methyl-BH4 (Table 2), whose
−
1
that when 1 mmol L 7,8-dihydrobiopterin was added, the
production of L-Dopa was reduced by 57%, decreasing from
2
0
substituents were –NH2 and –CH3, respectively. There is no
report on 7,8-dihydrobiopterin inhibiting SrTH until now.
The binding constant (K) for BH4/BH2/BH2 with adding 10
−
1
−1
8
1 mg L to 35 mg L (Fig. 6B). This indicates that the
accumulation of 7,8-dihydrobiopterin can inhibit the
synthesis of L-Dopa during the progress of the enzyme
reaction, which is consistent with our previous speculation.
We also evaluated the effect of 7,8-dihydrobiopterin on TcTH.
−
1
μmol
L
BH4/PH2 and SrTH interaction at different
temperatures were obtained by analyzing the fluorescence
changes data recorded at 265 nm according to the previous
−
1
45
It is worth noting that when 1 mmol L 7,8-dihydrobiopterin
was added, the production of L-Dopa by TcTH was reduced by
study. As shown in the illustrations of Fig. S12–S15,† the
plot of 1/(F
0
− F) against 1/[Q] was observed to be linear,
confirming a one-to-one interaction between the two
−
1
−1
3
4%, decreasing from 37.39 mg L to 24.79 mg L , which
was lower than 57% of SrTH (Fig. S3†). This indicates that
the accumulation of 7,8-dihydrobiopterin can also inhibit the
synthesis of L-Dopa during the progress of the enzyme
reaction by TcTH, which is consistent with that by SrTH.
partners. The thermodynamics parameters were also
calculated by the binding constants between BH4/BH2/BH2
−
1
with adding 10 μmol L BH4/PH2 and SrTH systems at
different temperatures as shown in Table 1 and Fig. S12–
S15.† Thermodynamic parameters including the free energy
change (ΔG), the enthalpy change (ΔH) and the entropy
change (ΔS) for the interaction between BH4/BH2/BH2 with
Characterization of inhibition by BH2 on SrTH
−
1
To obtain pure protein, we tried to express all the genes
using pET28a in the E. coli expression system. Unfortunately,
we could not be able to obtain the active protein and a large
number of inclusion bodies would be formed during the
expression process as shown in Fig. S9.† In order to analyze
the characterization of SrTH, the crude enzyme was partially
purified via ion exchange chromatography. The specific
adding 10 μmol L
BH4/PH2 and SrTH are vital for
characterizing the binding forces involved in the BH4/BH2/
−
1
BH2 with adding 10 μmol L BH4/PH2–protein complex
formation. ΔH and ΔS are positive indicating that the
hydrophobic interaction is dominating acting forces in BH2/
PH2–SrTH complex formation, which is different from the
4
6
interactions between BH4 and SrTH. The negative enthalpy
(ΔH) and entropy (ΔS) values of the interaction of BH4 and
SrTH indicate that van der Waals interactions and hydrogen
−
1
enzyme activity of SrTH reached 144.80 nmol min (mg of
−
1
protein) at 37 °C in this study as shown in Table S4 and
Fig. S10.† In this study, we immediately determined the
optimal catalytic temperature and pH of SrTH. We found
that over 50% specific enzyme activity of SrTH can be
detected at pH from 5 to 7.4 and at temperature from 20 °C
and 70 °C as shown in Fig. S11,† which indicates that SrTH
has better catalytic ability at a wider temperature range than
4
6
bonds played major roles in the reaction. It is worth noting
that ΔH and ΔS of the interaction of BH2 and SrTH with
−
1
adding 10 μmol L
BH4 in the solution changes into
negative, which indicates that van der Waals interactions and
hydrogen bonds are formed between BH2 and SrTH in the
presence of BH4. This may due to that the binding of BH4 to
SrTH can change the conformation of the protein and the
2
0,40
that from animals.
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Fig.
6 Inhibition analysis of enzyme reaction by 6,7-dihydrobiopterin and 7,8-dihydrobiopterin. A L-Dopa production with adding
−
1
−1
6
,7-dihydrobiopterin. 0.64 mmol L q-BH2 was added into the reaction ( ) with only adding 1 mmol L 2,6-dichloroindophenol as control (■); B
−
1
L-Dopa production with adding 7,8-dihydrobiopterin. 1 mmol L BH2 was added into the reaction ( ) with adding equal volume of ddH
2
O as
control (■). C Double reciprocal plot with taking 1/[S] as X axis and 1/V as Y axis. The concentrations of 7,8-dihydrobiopterin were 0 (■), 0.15 ( ),
−
1
0
(
.2 ( ), and 0.3 mmol L
(
); D double reciprocal plot with taking 1/[P] as X axis and 1/V as Y axis. The concentrations of BH4 were 0.05 (■), 0.1
−1
−1
−1
−1
2+
), 0.15 ( ), 0.2 ( ), 0.25 mmol L
(
). In the reaction, we supplemented with 1.5 g L tyrosine, 5 g L ascorbic acid, 100 μmol L Fe and 1 g
−1
−1
−1
L
crude enzyme powder containing SrTH. 1 g L crude enzyme powder containing SrTH and 0.25 mmol L BH4 were supplemented in the
−
1
−1
solution per 1 hour in A and B. 0.05–0.25 mmol L BH4 and 0–0.3 mmol L 7,8-dihydrobiopterin were supplemented in the enzyme reaction of C
and D. After catalyst at 37 °C 180 rpm, the production of L-Dopa was detected by HPLC. The dash lines indicate the feed points. All assays were
performed in triplicate.
Table 1 Binding parameters and thermodynamic parameters for the binding of various pterins to SrTH
4
−1
a
−1
−1 −1
−1
b
System
T (K)
K
a
(10 L mol
)
R
ΔG (kJ mol
)
ΔS (J mol
−53.97
K
)
ΔH (kJ mol
−49.02
)
R
SrTH (BH4)
303
42.22
27.63
20.09
18.53
27.59
50.19
33.15
3.710
1.205
16.90
33.54
95.87
0.9929
0.9966
0.9957
0.9996
0.9986
0.9978
0.9924
0.9970
0.9937
0.9950
0.9963
0.9907
−32.93
−32.66
−32.28
−30.04
−31.62
−33.83
−35.01
−31.88
−27.51
−29.27
−31.90
−35.59
0.9994
0.9991
0.9941
0.9968
3
3
10
15
SrTH (BH2)
298
315.48
−624.6
526.16
64.01
−221.2
127.60
3
3
03
10
c
SrTH (BH4&BH2)
303
3
3
10
15
SrTH (PH2)
298
3
3
03
10
a
b
c
−1
R is the correlation coefficient for the K
a
values. R is the correlation coefficient for the van't Hoff plot. 10 μmol L BH4 was added into
the solution before detecting the binding parameter of BH2.
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Table 2 Kinetic constants for tetrahydropterins in the reaction catalyzed by TH
−1
−1
Tetrahydropterin
Enzymes
K
m
(μM)
V
max (nmol mg min
)
Ref.
6(R)-BH4
6(S)-BH4
6-MPH4
6(R)-BH4
hTH1
hTH1
hTH1
SrTH
27.4 ± 6.3
218 ± 26
35.9 ± 4.7
224 ± 19
1146 ± 330
492 ± 7
720 ± 79
20
20
20
1108 ± 120
This study
The values represent the mean with the standard errors calculated from 3 experiments.
−
1
changed protein conformation can form van der Waals
interactions and hydrogen bonds with BH2. In order to
further explain the effect of hydrogen bonds between BH2
and SrTH, an appropriate amount of 7,8-dihydrobiopterin
was added to the reaction to systematically analyze the
influence of 7,8-dihydrobiopterin on the activity of SrTH. We
and 1 mmol L NADH at 25 °C for 1 h in HEPES-NaOH (pH
−1
7.4) buffer (Fig. S16†). In the enzyme reaction, 1 mmo L
NADH was almost completely converted into NAD (+), and the
content of BH4 produced by EcDHPR was 58.52 μmol L in
the solution at 1 h. In this study, we attempted to use BH4
regeneration by EcDHPR to alleviate BH2 inhibition of
L-tyrosine hydroxylation by crude SrTH. During the reaction, we
added 3 μg of EcDHPR and 1 mmol L NADH per 1 h and
found that L-Dopa could be produced continuously after 3 h
and improved to 0.17 g L at 6 h (Fig. 7C), which indicated
that the BH4 regeneration pathway can alleviate the inhibition
by BH2 during the reaction.
−1
found that the slope rate indicating K /V
of the four
m
max
−1
straight lines in the double reciprocal plot is the same and
was 0.19647, 0.20043, 0.20442 and 0.20268 after adding 0,
−
1
−1
0
.15, 0.2, and 0.3 mmol L 7,8-dihydrobiopterin, respectively,
into the reaction catalyzed by SrTH (Fig. 6C). The K and
Vmax values decreased, and K /V remained constant as the
m
m
max
7,8-dihydrobiopterin content increased (Fig. 6D), which
indicates that the inhibitory effect of 7,8-dihydrobiopterin on
SrTH is anticompetitive inhibition. In this study, we got the
kinetic constants for tetrahydropterins in the reaction
Discussion
To date, the tyrosine hydroxylases studied are derived from
cows, rats and humans.
2
1,35,39
catalyzed by SrTH and K
i
of 7,8-dihydrobiopterin on SrTH,
The actual titer of L-DOPA was
−
1
−1
which is 224 μM (K ), 1108 nmol mg min (Vmax), and 98
not quantified, even though a new pathway had been
demonstrated to hydroxylate L-tyrosine for producing L-Dopa
by TH in E. coli. BH4 is replaced by tetrahydromonapterin
(MH4) as the cofactor in a previous work. In this study, we
expressed tyrosine hydroxylases and sepiapterin reductases
selected from microorganisms in B. licheniformis and
successfully screened several TH/SPR synthesis systems that
can hydroxylate L-tyrosine for synthesizing L-Dopa (Fig. 2),
m
μM (K
i
) as shown in Tables 2 and 3. It is worth noting that
constant between crude
there is no obvious difference in K
i
2
5
SrTH and SrTH partially purified via ion exchange
chromatography as shown in Table S5.†
Effect of pure EcDHPR addition on the reaction catalyzed by
SrTH
which provides
a
novel foundation for L-tyrosine
The gene nfsB, encoding EcDHPR, was cloned into pET28a and
expressed in Bl-21 (DE3) at 16 °C and 200 rpm for 24 h with the
hydroxylation by TH in the future. Simultaneously, we found
anticompetitive inhibition of the SrTH-catalyzed reaction by
7,8-dihydrobiopterin, which has enriched theoretical support
for subsequent research on the rational and semirational
modification of SrTH and the biosynthesis of L-Dopa by the
TH/SPR synthesis system in B. licheniformis.
−1
addition of 0.1 mmol L IPTG. The product of the gene nfsB
was analyzed by SDS-PAGE using 12% polyacrylamide gel,
which indicated that the soluble expression of EcDHPR was
successfully achieved (Fig. 7A). Subsequently, EcDHPR was
purified using a HisTrap affinity column (Ni-NTA), and the
pure protein had a yellow color, which is consistent with
L-Tyrosine hydroxylation by TH requires the participation
of the cofactor BH4, which can be produced by GCHI, PTPS
and SPR from GTP in the folate biosynthesis pathway (http://
38
previous research (Fig. 7B). We detected the synthesis of BH4
−1
−1
−1
by HPLC using 0.1 g L pure EcDHPR, 0.5 mmol L q-BH2
www.kegg.jp/kegg/). This reaction could generate 23 mg L
Table 3
i
K values and type of inhibition for inhibitory or weakly binding pterins in the reaction catalyzed by TH
Pterins
Enzymes
K
i
(μM)
Inhibition
References
4
5
8
-Amino-BH4
-Methyl-BH4
-M-6,7-DMPH4
hTH1
hTH1
hTH1
SrTH
SrTH
16
63
617
Competitive vs. 6(R)-BH4
Competitive vs. 6(R)-BH4
Competitive vs. 6(R)-BH4
No inhibition
20
20
20
q-BH2
BH2
This study
This study
98 ± 16
Anticompetitive vs. 6(R)-BH4
The values represent the mean with the standard errors calculated from 3 experiments.
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BH4 in one step. The synthesis of L-Dopa indicates that BH4
can be successfully produced in B. licheniformis when PdSPR
from Photobacterium damselae is overexpressed. However, we
did not detect the production of BH4 in this article given that
only SPR was expressed in B. licheniformis. Further metabolic
engineering efforts are considered to analyze the synthesis of
BH4 after increasing GTP synthesis and enhancing the
expression of GCHI and PTPS in B. licheniformis, which will
provide a novel foundation for the metabolic synthesis of
L-Dopa in B. licheniformis.
The currently
hydroxylation include HpaBC and tyrosinase.
reported
enzymes for
L-tyrosine
There are
1
3,18
excellent works on the production of L-Dopa by HpaBC,
−
1
which can generate up to 0.69 g L L-Dopa from glucose in
1
1,13,14
shake flasks.
However, a large amount of melanin and
tyrosine remaining in the fermentation broth causes loss of
carbon flux to L-Dopa and its downstream high-value
products, such as hydroxytyrosol, plant alkaloids, betalain
4
–8,25
pigments and monolignols.
Tyrosine hydroxylase does
not have diphenolase (DP) activity, which is a good candidate
25
to overcome this challenge. Although the synthesis of
L-Dopa was successfully achieved by a combined whole-cell
catalyst in B. licheniformis, the titer of L-Dopa produced by
L-tyrosine hydroxylation was lower than those of the other
two enzymes in this study. There are four reasons that could
explain this phenomenon. First, L-Dopa is easily oxidized to
dopaquinone in the air and eventually becomes melanin
Fig. 7 Effect of pure EcDHPR addition on the reaction catalyzed by
SrTH. Protein content was quantified by the Bradford assay using
bovine serum albumin (BSA) as standard. 10 μg protein was analyzed
by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using 12%
polyacrylamide gel. Protein bands were stained by Coomassie Brilliant
Blue G250. A SDS-PAGE analysis of crude enzyme containing EcDHPR.
(
Fig. 2). Second, the production of BH4, an important
cofactor during L-tyrosine hydroxylation by TH, is low in
recombinant B. licheniformis expressing only PdSPR. During
L-tryptophan hydroxylation to produce 5-hydroxytryptophan,
both the synthesis and regeneration pathways of BH4 are
strengthened, and the titer of 5-hydroxytryptophan can reach
(
M) Molecular weight marker, (lane 1) E. coli Bl-21 (blank) and (lane 2)
E. coli Bl-21 (EcDHPR); B SDS-PAGE analysis of pure enzyme EcDHPR.
M) Molecular weight marker, (lane 1) pure EcDHPR and (lane 2) color
−
1 23,24
1
O
.61 g L .
At the same time, BH4 is easily oxidized by
(
3
6
.
These results indicate that BH4 is a limiting factor
of EcDHPR; C production of L-Dopa with adding pure EcDHPR and
2
−
1
−1
−1
NADH. In the reaction, we supplemented with 1.5 g L tyrosine, 5 g L
during the hydroxylation of L-tyrosine by B. licheniformis.
Unexpectedly, this system still cannot produce L-Dopa
continuously and effectively when adding sufficient BH4 and
crude SrTH protein during the catalysis process (Fig. 3B)
because the hydroxylation of L-tyrosine by SrTH was inhibited
by 7,8-dihydrobiopterin during the enzyme reaction (Fig. 6).
−
1
2+
ascorbic acid and 100 μmol L Fe at a time. At the same time, 1 g L
crude enzyme powder containing SrTH and 0.25 mmol L BH4 were
−
1
supplemented with per 1 hour. In the sample ( ), we added 3 μg
−
1
EcDHPR and 1 mmol L NADH per 1 hour. And in the control (■), we
added the same volume of HEPES-NaOH (pH 7.4). After catalyst at 37
°C 180 rpm for 6 h, L-Dopa in the supernatant was detected by HPLC.
We compared the K
i
among the four different pterin
compounds and found that inhibitory effect of BH2 (K 98
i
μM) on TH is weaker than 4-amino-BH4 (K
i
16 μM) and
BH4 when folE from E. coli and PTPS and SPR from rats were
coexpressed in E. coli. Compared with folE from E. coli, the
expression of GCHI encoded by the mtrA gene derived from
Bacillus subtilis in E. coli can enhance the production of
5-methyl-BH4 (K 63 μM) but stronger than 8-M-6,7-DMPH4
i
(Ki 617 μM) as shown in Table 3. In this study, it was
demonstrated that the BH4 regeneration pathway can not
only provide more cofactor BH4 but also alleviate the
anticompetitive inhibition of L-tyrosine hydroxylation by
7,8-dihydrobiopterin (Fig. 7C). Fourth, tyrosine hydroxylase
can be affected by L-tyrosine (over 100 μM) and oxygen
24
5
-hydroxytryptophan.
This was because when folE was
substituted by GCHI from B. subtilis, BH4 productivity was
2
2
improved 1.5-fold. We analyzed the BH4 synthesis pathway
of B. licheniformis by KEGG and found that it contained a
complete synthetic pathway of the BH4 precursor
2
1
(higher than 4.8%) in the catalytic reaction. Furthermore,
SrTH lacks the N-terminal domain as a eukaryotic enzyme,
which has the function of physically blocking the entrance to
6
-pyruvoyltetrahydropterin, in which GCHI has been proven
2
2
1
to be functional. Therefore, we proposed a speculation that
the active site in the substrate-free enzyme. The reversible
B. licheniformis overexpressing exogenous SPR can synthesize
phosphorylation of serine residues in the N-terminal domain
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plays an important role in decreasing the affinity for References
4
1,42
L-Dopa.
At the same time, the lack of an N-terminal
domain may cause structural instability of the SrTH
1 P. F. Fitzpatrick, Biochemistry, 2003, 42, 14083–14091.
2 P. R. Dunkley and P. W. Dickson, J. Neurochem., 2019, 149,
706–728.
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3
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pterin, specifically with the N-8 and O-4 positions of pterin,
3
5
respectively. However, the two positions of SrTH were
substituted by Val126 and Phe203, which may decrease the
affinity for BH4. Next, site saturation mutagenesis will be
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SrTH to research the influence on BH4 binding.
It will be a huge challenge to efficiently synthesize L-Dopa
by hydroxylating L-tyrosine in B. licheniformis using the TH/
SPR dual-enzyme synthesis system. In the future, we will
focus on two research objectives regarding L-tyrosine
hydroxylation by B. licheniformis harboring SrTH. Firstly, we
will try to increase the expression of SrTH in B. licheniformis.
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2
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by many factors, including but not limited to tyrosine
hydroxylase, BH4, L-tyrosine and oxygen. In the enzymatic
catalysis process, only ensuring sufficient enzyme and substrate
cannot continuously and efficiently generate L-Dopa.
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7
regeneration pathway. Further studies should be considered to
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Acknowledgements
This work was supported by National Key Research &
Development
Program
of
China
(2020YFA0907700,
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Discipline Program of Light Industry Technology and
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Project of Jiangsu Higher Education Institutions and the
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