Gene cloning and characterization of thiourocanate hydratase from Burkholderia sp. HME13

Article abstract of DOI:10.1093/jb/mvz098

A novel enzyme, thiourocanate hydratase, which catalyses the conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp. HME13. When the HME13 cells were cultured in medium containing ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity was detected in the crude extract from the cells. However, activity was not detected in the crude extract from HME13 cells that were cultured in Luria-Bertani medium. The gene encoding thiourocanate hydratase was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and 55°C and was stable between pH 5.0 and 10.5, and at temperatures up to 45°C. The K_m and V_max values of thiourocanate hydratase towards thiourocanic acid were 30 μM and 7.1 μmol/min/mg, respectively. The enzyme was strongly inhibited by CuCl₂ and HgCl₂. The amino acid sequence of the enzyme showed 46% identity to urocanase from Pseudomonas putida, but thiourocanate hydratase had no urocanase activity.

Full text of DOI:10.1093/jb/mvz098

Regular paper
Biochemistry (Enzymology)
Gene cloning and characterization of thiourocanate hydratase from
Burkholderia sp. HME13.
Hisashi MURAMATSU,1† Haruna MIYAOKU,2 Syuya KURITA,2 Hidenori
MATSUO,3 Takehiro KASHIWAGI,1 Chul-Sa KIM,1 Motoko HAYASHI,4 Hiroaki
YAMAMOTO,4 Shin-ichiro KATO1 and Shinji NAGATA1
1Multidisciplinary Science Cluster, Research and Education Faculty, Kochi
University, B200 Monobe, Nankoku, Kochi 783-8502, Japan.
2Department of Agriculture, Kochi University, B200 Monobe, Nankoku, Kochi
783-8502, Japan.
3Graduate School of Integrated Arts and Sciences, Kochi University, B200
Monobe, Nankoku, Kochi 783-8502, Japan.
4Corporate Research Center, R&D Headquaters Management, Daicel
Corporation 1-1 Shinko-cho, Myoko-shi, Niigata 944-8550, Japan
Running title: Thiourocanate hydratase from Burkholderia sp. HME13.
†To whom correspondence should be addressed.
Phone: +81-88-864-5187; Fax: +81-88-864-5197
E-mail: hmura@kochi-u.ac.jp
© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights
reserved.
1

Note: Nucleotide sequence data reported are available in the
DDBJ/EMBL/GenBank databases under the accession number LC481535.
2

SUMMARY
A novel enzyme, thiourocanate hydratase, which catalyzes the
conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic
acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp.
HME13. When the HME13 cells were cultured in medium containing
ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity
was detected in the crude extract from the cells. However, activity was not
detected in the crude extract from HME13 cells that were cultured in
Luria-Bertani (LB) medium. The gene encoding thiourocanate hydratase was
cloned and expressed in Escherichia coli, and the recombinant enzyme was
purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and
55 °C, and was stable between pH 5.0 and 10.5, and at temperatures up to
45 °C. The Km and Vmax values of thiourocanate hydratase toward thiourocanic
acid were 30 M and 7.1 mol/min/mg, respectively. The enzyme was strongly
inhibited by CuCl2 and HgCl2. The amino acid sequence of the enzyme showed
46% identity to urocanase from Pseudomonas putida, but thiourocanate
hydratase had no urocanase activity.
Keywords: ergothioneine; thiourocanic acid; thiourocanate hydratase;
urocanase; Burkholdria
3

Ergothioneine, a water-soluble naturally occurring amino acid analog,
is a derivative of histidine betaine that contains a thiol group attached to the C2
atom of the imidazole ring. In solution, ergothioneine exists as a tautomer
between the thiol and thione forms, and at physiological pH, it exists
predominantly as the thione. (1) Ergothioneine is a strong antioxidant for
hydroxyl radicals, hypochlorous acid, and peroxynitrite, (2, 3) and has been
shown to be effective in preventing oxidative damage in cells. Ergothioneine is
found in many living organisms (4-12), but is synthesized by only a few
organisms, such as certain microbes and mushrooms. (13-18) Mammalian cells
cannot synthesize ergothioneine and must absorb ergothioneine from dietary
sources. It is believed that ergothioneine synthesized by microbial organisms is
probably incorporated into other organisms, such as plants and animals.
The microbial ergothioneine biosynthetic pathway, which is the source
of ergothioneine for higher organisms, has been much investigated. (7, 17) In
contrast, the ergothioneine utilization pathway is poorly understood.
Ergothionase, which catalyzes the reaction from ergothioneine to thiourocanic
acid and trimethylamine, is the only enzyme in this pathway that has been
reported to date. (19, 20) Hence, we sought to identify the enzyme acting on
thiourocanic acid in the Burkholderia sp. HME13, which is isolated from the soil.
We have reported that Burkholderia sp. HME13 cells cultivated in a medium
containing ergothoneine as the sole nitrogen source expressed ergothionase.
(20) The gene encoding ergothionase from Burkholderia sp. HME13 has been
identified and the molecular characteristics of the recombinant enzyme have
been reported. (20) We analyzed the nucleotide sequence of the upstream
region of the ergothionase gene from Burkholderia sp. HME13 and identified the
gene encoding the protein, which showed similarity to urocanases. Herein, we
demonstrate that the gene product, which showed similarity to urocanases,
4

showed thiourocanate hydratase activity and report the characterization of this
enzyme (Scheme 1).
5

Materials and Methods
Materials
Ergothioneine and thiourocanic acid were a gift from Daicel
Corporation (Osaka, Japan). Polypepton was from Nihon Pharmaceutical
(Tokyo, Japan). Yeast extract and molecular-weight marker-proteins for gel
filtration were from Oriental Yeast (Tokyo, Japan). Restriction enzymes and kits
for genetic manipulation were from Takara Bio (Kyoto, Japan), Toyobo (Osaka,
Japan), New England Biolabs (Beverly, MA, USA), Nippon gene (Tokyo, Japan),
Bioneer (Alameda, CA, USA), Roche (Indianapolis, IN, USA), and Stratagene
(La Jolla, CA, USA). All other reagents were of analytical grade and were
obtained from Sigma (St. Louis, MO, USA), Nacalai Tesque (Kyoto, Japan), and
Wako Pure Chemical Industries (Osaka, Japan). Toyopearl DEAE-650M,
Toyopearl Butyl-650M, and Toyopearl AF-Blue HC-650M were purchased from
Tosoh (Tokyo, Japan). Superose 12 HR 10/30 was obtained from Amersham
Pharmacia Biotech (Uppsala, Sweden).
Microorganisms and culture conditions
Burkhorderia sp. HME13 was cultivated at 30 °C in LB medium or
ergothioneine medium containing 0.023% ergothioneine, 0.5% glucose, 1.0%
KH2PO4, 0.2% citric acid, and 0.02% MgSO4·7H2O (pH 7.2) with reciprocal
shaking. Burkhorderia sp. HME13 was cultivated in LB medium for 20 h, or
ergothioneine medium for 45 h. The cells were harvested by centrifugation,
washed twice with 0.85% NaCl, and stored at –80 °C until use.
Escherichia coli was cultivated at 37 °C overnight in LB medium on a
reciprocal shaker. Recombinant E. coli cells were grown aerobically at 37 °C for
14 h in LB medium containing 0.1 mg/ml ampicillin. The culture was
supplemented with 1 mM isopropyl--D-thiogalactopyranoside (IPTG) and
6

grown for a further 3 h. The cells were harvested by centrifugation, washed
twice with 0.85% NaCl, and stored at –80 °C until use.
Enzyme assay
The washed bacterial cells were resuspended in 20 mM potassium
phosphate buffer (pH 7.2) containing 1 mM phenylmethylsulfonyl fluoride
(PMSF), 0.01% 2-mercaptoethanol (2-ME), 20% glycerol, and 50 mM NaCl and
disrupted by sonication on ice. The resulting crude extract was centrifuged at
4 °C and the supernatant was used for the enzyme assay.
Thiourocanate hydratase activity was assayed by monitoring the
decrease in absorbance at 311 nm, caused by the consumption of thiourocanic
acid, at 30 °C in a 1-ml standard reaction mixture containing 80 M thiourocanic
acid and 0.1 M potassium phosphate buffer (pH 7.2). One unit of the enzyme
was defined as the amount that catalyzed the consumption of 1 mol of
thiourocanic acid per minute in the reaction.
Ergothionase assays were performed according to the method of John
B. Wolff (19). Ergothionase activity was assayed by monitoring the increase in
the absorbance at 311 nm, caused by the production of thiourocanic acid, at
30 °C in a 1-ml standard reaction mixture containing 0.25 mM ergothioneine
and 10 mM sodium pyrophosphate buffer (pH 9.0). One unit of the enzyme was
defined as the amount that catalyzed the formation of 1 mol of thiourocanic
acid per minute in the reaction.
The concentration of thiourocanic acid was determined using the
following molar extinction coefficients at 311 nm: 18,000 M-1cm-1 (10 mM
sodium pyrophosphate buffer, pH 8.0–9.0 at 30 °C), 18,000 M-1cm-1 (10 mM
sodium phosphate buffer, pH 7.2 at 20–80 °C), 18,000 M-1cm-1 (0.1 M
potassium phosphate buffer, pH 7.2 at 20–80 °C), 19,000 M-1cm-1 (0.1 M
sodium acetate buffer, pH 5.0–6.5 at 30 °C), 18,000 M-1cm-1 (0.1 M sodium
7

phosphate buffer, pH 6.5–7.5 at 30 °C), 18,000 M-1cm-1 (0.1 M potassium
phosphate buffer, pH 6.5–7.5 at 30 °C), 18,000 M-1cm-1 (0.1 M HEPES buffer,
pH 7.0–8.0 at 30 °C), 19,000 M-1cm-1 (0.1 M Tris-HCl buffer, pH 7.5–8.5 at
30 °C), 18,000 M-1cm-1 (0.1 M glycine NaOH buffer, pH 8.5–9.5 at 30 °C),
17,000 M-1cm-1 (0.1 M glycine NaOH buffer, pH 10.0 at 30 °C), 15,000 M-1cm-1
(0.1 M glycine NaOH buffer, pH 10.5 at 30 °C), and 14,000 M-1cm-1 (0.1 M
glycine NaOH buffer, pH 11.0 at 30 °C). (20)
Urocanase activity was assayed by monitoring the decrease in
absorbance at 277 nm, caused by the consumption of urocanic acid, at 30 °C in
a 1-ml standard reaction mixture containing 80 M urocanic acid and 0.1 M
potassium phosphate buffer (pH 7.2). One unit of the enzyme was defined as
the amount that catalyzed the consumption of 1 mol of urocanic acid per
minute in the reaction. The molar extinction coefficient at 277 nm of urocanic
acid used in the calculation was 18,800 M-1cm-1.
Sequencing of the DNA fragment containing ergothionase gene and the
upstream region of this gene
Genomic DNA was prepared from Burkholderia sp. HME13 with an
UltraClearn Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA,
USA). Based on the genome sequences of B. pseudomallei 1106a, B.
pseudomallei K96243, B. pseudomallei 1710b, and Burkholderia sp. 383, PCR
primers (Primer 1: 5’-ATTCCGCTGTCGTCGTCGGTGTTCGATCC-3’ and
Primer 2: 5’-CCCGACGCGAGCTGCACGAGATGCTT-3’) were designed for
amplification of the DNA fragment containing the ergothionase gene and the
upstream region of this gene. The DNA fragment was amplified by PCR in a
50-l reaction mixture containing 1 Ex Taq buffer, 0.2 mM deoxynucleotide
triphosphates, 10 pmol of each primer, 140 ng of Burkholderia sp. HME13
genomic DNA, and 1.3 units of Ex Taq DNA polymerase. PCR was performed
8

as follows: preincubation at 94 °C for 1 min and then 30 cycles of 98 °C for 10 s
and 68 °C for 3 min, and finally 72 °C for 10 min. PCR products were
sequenced with an ABI PRISM 3100-Avant Genetic Analyzer and a DNA
sequencing kit (BigDye Terminator v3.1 Cycle Sequencing Kit, Foster City, CA,
USA).
Cloning of the gene encoding thiourocanate hydratase
The gene encoding thiourocanate hydratase was amplified by PCR in
a 50-l reaction mixture containing 1 pfuUltra High-Fidelity DNA polymerase
reaction buffer, 0.2 mM deoxynucleotide triphosphates, 20 pmol of each primer
(5’-GGAATTCCATATGACCCGCACCTTCGAAGTCACG-3’
5’-GGGAAGCTTTTAAAGCCGGATATGCGGAATCTGC-3’),
and
of
290
ng
Burkholderia sp. HME13 genomic DNA, and 2.5 units of pfuUltra High-Fidelity
DNA polymerase. PCR was performed as follows: preincubation at 95 °C for 2
o
min and then 30 cycles of 95 °C for 30 s, 57 °C for 1 min and 72 C for 90 s,
o
and finally 72 C for 10 min. The PCR product was digested with NdeI and
HindIII and ligated into pSE420U, which had been previously digested with the
same restriction enzymes. The resultant plasmid was introduced into E. coli
JM109 to produce the recombinant thiourocanate hydratase.
Purification of recombinant thiourocanate hydratase from E. coli
The standard buffer used throughout the purification was 20 mM
potassium phosphate buffer (pH 7.2) containing 20% glycerol, and 50 mM NaCl.
Thiourocanate hydratase-expressing E. coli JM109 cells were suspended in the
standard buffer containing 1 mM PMSF and 0.01% 2-ME, and disrupted on ice
by sonication. Subsequent operations were performed at 4 °C. After
centrifugation, the enzyme solution was dialyzed against the standard buffer
containing 0.01% 2-ME and applied to a Toyopearl DEAE-650M column
9

equilibrated with the standard buffer, and eluted with a linear gradient of 0.05–
0.5 M NaCl in the buffer. The enzyme fractions were pooled, concentrated with
Vivaspin 20 (30,000 MWCO PES) ultrafiltration units (Sartorius Stedim Biotech
GmbH, Germany), and dialyzed against the standard buffer containing 35%
saturated ammonium sulfate and 0.01% 2-ME. The enzyme solution was then
applied to a Toyopearl Butyl-650M column equilibrated with the standard buffer
containing 35% saturated ammonium sulfate, and eluted with a linear gradient
of 35–0% saturated ammonium sulfate in the buffer. The enzyme fractions were
pooled, concentrated with Vivaspin 20 (30,000 MWCO PES) ultrafiltration units,
and dialyzed against the standard buffer containing 0.01% 2-ME. The enzyme
solution was then applied to a Toyopearl AF-Blue HC-650M column equilibrated
with the standard buffer, and eluted with a linear gradient of 0.05–1.0 M NaCl in
the buffer. The enzyme fractions were pooled, concentrated with Vivaspin 20
(30,000 MWCO PES) ultrafiltration units, and dialyzed against the standard
buffer. The recombinant enzyme was purified to homogeneity (Table 1, Fig. 1).
The final preparation of the enzyme was stored at –80 °C until use.
The N-terminal amino acid sequence of the purified enzyme was
determined by Edman degradation with a Procise 492 protein-sequencing
system (Applied Biosystems, Foster City, CA, USA). The N-terminal amino acid
sequence of the purified enzyme, MTRTFEVTSGNTLR, was identical to the
deduced amino acid sequence of the target gene product.
Determination of molecular mass of thiourocanate hydratase
The recombinant enzyme solution was applied to a Superdex 200
10/300 GL column equilibrated with 50 mM potassium phosphate buffer (pH
7.2) containing 0.2 M NaCl. The column was equipped with a BioLogic DuoFlow
Chromatography System (BIO-RAD, Hercules, CA, USA), and developed at
4 °C at a flow rate of 0.75 ml/min. Estimation of the molecular mass was
10

performed with the following protein markers: glutamate dehydrogenase (290
kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), adenylate kinase (32
kDa), and cytochrome (12.4 kDa).
Kinetic Analysis
The initial rates of the thiourocanate hydratase reactions were
measured with various concentrations of substrate. Data were fitted to the
hyperbolic Michaelis-Menten equation, and kinetic parameters were calculated
using nonlinear least-squares regression with Kaleida Graph software
(Adelbeck Software, Reading, PA, USA).
LC-MS and NMR analysis of the reaction product of thiourocanate hydratase
To analyze the reaction product of thiourocanate hydratase with
LC-MS, a 0.2-ml reaction mixture containing 2.0 mM thiourocanic acid, 20 mM
potassium phosphate buffer (pH 7.2), and 0.85 mg/ml purified enzyme was
incubated for 1 h at 30 °C. LC-MS data of the reaction product were acquired
using a tandem mass spectrometer (ACQUITY TQD, Waters, Milford, MA, USA)
coupled to an ultra-performance liquid chromatography (UPLC) system (ACQUITY
UPLC System, Waters, Milford, MA, USA) using a reversed-phase column (Inertsil
ODS-4 column  4.6 × 250 mm, GL Science, Tokyo, Japan) eluted with 20%
methanol in water containing 0.24% acetic acid. The flow rate was set at 0.5
ml/min. The mass spectrometer was operated in the positive and negative electrospray
ionization modes (ESI+ and ESI–), with a capillary voltage of 3 kV. The source and
desolvation temperatures were 150 and 350 °C, respectively.
To analyze the reaction product of the enzyme by NMR, a 60-ml
reaction mixture containing 2.0 mM thiourocanic acid, 20 mM potassium
phosphate buffer (pH 7.2), and 0.85 mg/ml thiourocanate hydratase was
incubated for 2 h at 30 °C. After adding 0.3 ml of formic acid, the resultant
11

mixture was applied to a Sep-Pak® Plus C18 cartridge (Waters, Milford, MA,
USA). The reaction product was eluted with methanol and dried with an
evaporator and lyophilizer. The reaction product was dissolved in methanol-d4.
1H and 13C NMR data were recorded by a JEOL JNM-ECX-500 spectrometer in
methanol-d4 with tetramethylsilane as an internal standard. The letters (br)s, d, t,
q, and m represent (broad)singlet, doublet, triplet, quartet, and multiplet,
respectively, and coupling constants are given in Hz.
Other analytical methods
Protein concentrations were determined by protein assay CBB
solution (Nacalai Tesque, Kyoto, Japan) with bovine serum albumin as a
standard. The purity and molecular mass of the subunit of thiourocanate
hydratase were examined by SDS-PAGE with the following protein markers
(Nacalai Tesque, Kyoto, Japan): myosin (200 kDa), -galactosidase (116 kDa),
bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31
kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa).
Gels were stained with Coomassie Brilliant Blue G-250. Nucleotide and amino
acid sequences were obtained from the database at National Center for
Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/). A similarity search
was performed with the BLAST program (21) at NCBI. Multiple alignments were
obtained with the ClustalW program (22) at GenomeNet (www.genome.ad.jp/).
Calculation of the molecular weight of the proteins was performed with the
compute
pI/Mw
tool
(23-25)
at
ExPASy
Proteomics
Server
(http://au.expasy.org/). Prediction of the cellular localization of proteins used
PSORTb version 3.0.2 (http://www.psort.org/psortb/) (26). Prediction of
transmembrane helices in the proteins was performed with TMHMM Server v.
2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (27).
12

Results
Identification of the gene encoding the thiourocanate-metabolizing enzyme
In silico analysis of genomic information from B. pseudomallei 1106a,
B. pseudomallei K96243, B. pseudomallei 1710b, and Burkholderia sp. 383
suggested that the urocanase homolog gene is located upstream of the
ergothionase gene. Thus, a DNA fragment of approximately 5.2 kb containing
the ergothionase gene and the upstream region of this gene was amplified and
the 5,135-bp nucleotide sequence was determined. The amplified DNA
fragment contained three genes, the ergothionase gene, the putative major
facilitator superfamily transporter gene, and a gene encoding a protein that
showed 46% amino acid sequence identity to urocanase from Pseudomonas
putida. (28) The DNA fragment containing the gene encoding the urocanase
homolog was amplified by PCR and ligated into pSE420U. E. coli JM109 cells
harboring the resultant plasmid were cultivated and the expression of
recombinant enzyme was induced by the addition of IPTG. The crude extract
from the recombinant E. coli JM109 cells was used in the thiourocanate
hydratase assay and a decrease in absorbance at 311 nm caused by the
consumption of thiourocanic acid was observed. These results indicated that
the recombinant enzyme had thiourocanate-metabolizing activity. To the best of
our knowledge, this enzyme acting on thiourocanic acid has not previously been
reported.
Ergothionase activity has been detected in the crude extract of
Burkholderia sp. HME13 cells cultivated in a medium containing erogothioneine
as the sole nitrogen source. (20) Thiourocanate-metabolizing activity was also
detected in the crude extract from Burkholderia sp. HME13 cells cultured in
ergothioneine medium (apparent specific activity: 0.58 mol/min/mg). However,
13

no thiourocanate-metabolizing activity was detected in the crude extracts from
the strain cultured in LB medium.
Determination of the reaction product of the thiourocanate-metabolizing enzyme
We theorized that the metabolic process for thiourocanic acid should
be similar to that for urocanic acid, and that thiourocanic acid would be
converted to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid (molecular
weight: 188.03) in Burkholderia sp. HME13 cells (Scheme 1). To test this
hypothesis, the recombinant enzyme acting on thiourocanic acid was purified to
homogeneity (Table 1, Fig. 1) and was used for LC-MS and NMR analyses of
the reaction product of the enzyme.
After incubation for 1 h at 30 °C, the peak corresponding to
thiourocanic acid (retention time: 14.6 min) was not detected in the reaction
mixture by LC-MS analysis, and a new peak (retention time: 10.1 min)
corresponding to the reaction product of the enzyme appeared. This peak had
several fragment ions derived from the expected product, including 170.92
[M+H–H2O]+, and 188.93 [M+H]+ in positive ESI mass analysis, and 186.96
[M-H]– and 375.04 [2M-H]– in negative ESI mass analysis. After isolation, the
reaction product was analyzed by NMR spectroscopy. The reaction product of
the
thiourocanate-metabolizing
enzyme
was
determined
to
be
3-(5-oxo-2-thioxoimidazolidin-4-yl) propanoic acid, as the 13C-NMR and
1H-NMR data agreed with the literature data. (29) These results suggested that
the thiourocanate-metabolizing enzyme was thiourocanate hydratase, which
converts thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propanoic acid.
3-(5-Oxo-2-thioxoimidazolidin-4-yl) propanoic acid LC-MS (ESI positive)
m/z (%): 188.93 (100.0) [M+H]+ and 170.92 (63.8) [M+H–H2O]+ (ESI negative) m/z
(%): 186.96 (100.0) [M-H]– and 375.04 (15.1) [2M-H]–. ¹H-NMR (methanol-d4) δH:
4.26 (1H, dd, J = 6.5 and 5.5, H-4’), 2.42 (2H, t, J = 7.0, H-2), 2.10 (1H, ddt, J = 14.0,
14

7.5 and 7.0, H-3a), 1.96 (1H, dq, J = 14.0 and 7.0, H-3b). ¹³C-NMR (methanol-d4) δC:
184.8 (s, C-2’), 178.1 (s, C-1), 176.1 (s, C-5), 61.7 (d, C-4’), 29.8 (t, C-2), 27.6 (t, C-3).
Urocanase assay using purified thiourocanate hydratase
Thiourocanate hydratase activity was detected in the reaction mixture
containing 80 M thiourocanic acid, 0.1 M potassium phosphate buffer (pH 7.2),
and 17 g/ml purified thiourocanate hydratase. However, urocanase activity
was not detected after addition of 17 or 170 g/ml of purified thiourocanate
hydratase to a reaction mixture containing 80 M urocanic acid and 0.1 M
potassium phosphate buffer (pH 7.2). In addition, urocanase activity was not
detected in the reaction mixture containing 0.8 mM urocanic acid, 0.1 M
potassium phosphate buffer (pH 7.2), and 0.85 mg/ml purified thiourocanate
hydratase. From these results, we concluded that thiourocanate hydratase has
no urocanase activity.
Subunit structure of thiourocanate hydratase
The molecular mass of the purified enzyme was determined by gel
filtration to be 72 kDa. The molecular mass of the subunit calculated from the
deduced amino acid sequence of the enzyme was 59824.19 Da. Therefore, the
enzyme is likely to be a monomeric protein.
Effect of pH and temperature on thiourocanate hydratase
The optimum pH of thiourocanate hydratase was analyzed using a
variety of 0.1 M buffer solutions at 30 °C (Fig. 2A). Potassium phosphate buffer
was suitable for the enzyme reaction and the enzyme showed a maximum
activity at pH 7.5 in this buffer. A marked difference in activity between using
potassium phosphate buffer and Tris-HCl buffer at pH 7.5 was observed.
Tris-HCl solution (pH 7.5) was added to a reaction mixture containing 80 M
15

thiourocanic acid, 0.1 M potassium phosphate buffer (pH 7.5), and the purified
enzyme, then the enzyme assay was performed. Tris-HCl at concentrations of
20 mM, 50 mM, and 0.1 M inhibited the enzymatic reaction by 46%, 65%, and
78%, respectively. Similarly, a marked difference in activity between using
potassium phosphate buffer and sodium acetate buffer at pH 6.5 was observed.
Sodium acetate at concentrations of 20 mM, 50 mM, and 0.1 M inhibited the
enzymatic reaction by 69%, 86%, and 96%, respectively. The optimum
temperature of the enzyme was determined by measuring the activity in a
standard reaction mixture, and varying the temperature from 25 to 75 °C. The
enzyme showed maximum activity at 55 °C, and more than 75% of the
maximum activity was retained between 45 °C and 65 °C. (Fig. 2B) The pH
stability of the enzyme was examined by incubating the enzyme for 1 h at 30 °C
in various buffers prior to assay in the standard reaction mixture at 30 °C. More
than 85% of the enzyme activity of the untreated thiourocanate hydratase was
retained between pH 5.0 and pH 10.5. (Fig. 2C) After incubation of the enzyme
at different temperatures for 1 h, the enzyme activity was measured in the
standard reaction mixture at 30 °C. The enzyme was stable up to 45 °C and lost
activity at 65 °C. (Fig. 2D)
Kinetic analysis
The Km and Vmax values were determined by steady-state kinetic
analysis using purified thiourocanate hydratase. The data obtained gave a good
global fit to the Michaelis-Menten equation. The Km and Vmax values of
thiourocanate hydratase for thiourocanic acid were 30 M and 7.1 mol/min/mg,
respectively.
Effect of various ions and compounds on thiourocanate hydratase
16

CuCl2 and HgCl2 inhibited the enzymatic reaction by 95.6% and 99%,
respectively (Table 2). RbCl, CaCl2, NiCl2, CoCl2, ZnCl2, and BaCl2 had little
effect
2-mercapto-4-methylimidazole, -N-methyl-L-thiohistidine, L-ergothioneine,
D-ergothioneine, 2-imidazolidinethione, L-2-thiohistidine, α-N,
on
the
reaction.
The
molecular
structures
of
N’-dimethyl-L-2-thiohistidine, and urocanic acid are similar to the structure of
thiourocanic acid. 2-Mercapto-4-methylimidazole had no effect on the
thiourocanate hydratase reaction (Table 3). α-N-Methyl-L-thiohistidine,
D-ergothioneine, L-ergothioneine, and 2-imidazolidinethione inhibited the
enzymatic
reaction
by
less
than
5%.
L-2-Thiohistidine,
α-N,
N’-dimethyl-L-2-thiohistidine and urocanic acid inhibited the reaction by 11%–
19%. PMSF and glyoxal sodium bisulfite inhibited the reaction by 25% and 37%,
respectively.
Discussion
After incubation of the reaction mixture containing thiourocanic acid,
potassium phosphate buffer, and purified thiourocanate hydratase for 1 h at
30 °C, the peak corresponding to thiourocanic acid was not detected in the
reaction mixture by LC-MS analysis. Therefore, we believe that the conversion
rate of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid by
thiourocanate hydratase is faster than the reverse reaction by the enzyme.
The amino acid sequence of thiourocanate hydratase from
Burkholderia sp. HME13 showed 46% identity to urocanase from Pseudomonas
putida. The crystal structure of urocanase from P. putida has been resolved
(PDB ID: 1UWK, 1UWL and 1W1U). Urocanase contains a tightly bound NAD+
molecule that acts as an electrophile (30). NAD+ binds via the pyrophosphate
17

moiety at the typical glycine-rich sequence fingerprint at position 174–179, and
the adenine ribose unit interacts with Glu197 of urocanase. This is the
commonly observed binding mode of NAD+ in Rossmann-type chain folds.
These glycine and glutamate residues were conserved in thiourocanate
hydratase (Fig. 3). Therefore, thiourocanate hydratase is also likely to contain a
NAD+ molecule that acts as an electrophile.
The putative metabolic process for converting ergothioneine to
3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid is quite similar to L-histidine
degradation by the histidine utilization (Hut) pathway (Fig. 4) (31). Assuming
that thiourocanate hydratase is involved in the utilization of ergothioneine in
Burkholderia sp. HME13, 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid may
be metabolized to glutamic acid by process similar to the Hut pathway. We have
reported that ergothionase from Burkholderia sp. HME13 contains the signal
sequence on its N-terminus. (20) PSORTb version 3.0.2 predicted that
ergothionase may localize in the periplasm or the cytoplasmic membrane, and
TMHMM Server v. 2.0 also predicted that ergothionase has no transmembrane
helices. From these bioinfomatic analyses, we believe that erogothionase
probably localizes in the periplasm. Moreover, PSORTb version 3.0.2 predicted
that thiourocanate hydratase is localized in the cytosol. These results indicate
that Burkholderia sp. HME13 may convert ergothioneine into thiourocanic acid
by ergothionase in the periplasm, and then, the thiourocanic acid may be
transported to the cytosol and hydrated. We believe that it is possible that the
expression of ergothioneine utilization genes may be regulated by the
thiourocanic acid-mediated system. The 3-(5-oxo-2-thioxoimidazolidin-4-yl)
propionic acid produced by thiourocanate hydratase may be metabolized by
several enzyme reactions in the cytosol.
18

Funding
This work was supported by JSPS KAKENHI, Grant-in-Aid for Young
Scientists (B), Grant Number JP25850052.
Acknowledgement
We thank the ‘National BioResource Project (NIG, Japan): E. coli’ for
the gift of E. coli JM109 strain. Victoria Muir, PhD, from Edanz Group
(www.edanzediting.com/ac) edited a draft of this manuscript.
Conflict of interest
None declared.
19

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23

Table 1. Purification of the thiourocanate-metabolizing enzyme.
Steps
Activity
units
Protein Specific activity Yield
Purity
-fold
1.0
mg
1,600
680
units/mg
2.5
%
100
77
Crude extract
3,900
Toyopearl–DEAE 650M 3,000
Toyopearl–Butyl 650M 2,500
Toyopearl–AF–Blue 650M 1,200
4.5
1.8
380
6.5
62
2.6
170
7.2
31
2.9
1

Table 2. Effect of various ions on the activity of thiourocanate hydratase.
The enzyme activity was determined in a reaction mixture containing 80 M
thiourocanic acid, 0.1 M potassium phosphate buffer (pH 7.2), 17 g/ml
thiourocanate hydratase, and the various ions at 30 °C. The data were obtained
from a single experiment.
Compounds
None
Concentration (mM)
Relative activity (%)
—
100
100
100
97
NaCl
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
KCl
RbCl
CsCl
100
100
97
MgSO4
CaCl2
NiCl2
98
CoCl2
CuCl2
ZnCl2
BaCl2
HgCl2
96
4.4
94
94
1.0
1

Table 3. Effect of various compounds on the activity of thiourocanate hydratase.
The enzyme activity was determined in a reaction mixture containing 80 M
thiourocanic acid, 0.1 M potassium phosphate buffer (pH 7.2), 17 g/ml
thiourocanate hydratase, and the various compounds at 30 °C. The data were
obtained from a single experiment.
Compounds
Concentration (mM)
Relative activity (%)
None
—
100
100
100
100
98
98
96
96
96
95
95
95
94
94
91
89
87
81
75
63
Diethyldithiocarbamate
1.0
2-Mercapto-4-methylimidazole
Ethylenediaminetetraacetic acid
-N-Methyl-L-thiohistidine
D-Ergothioneine
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
L-Ergothioneine
Aminoguanidine
DL-Penicillamine
2-Imidazolidinethione
Semicarbazide
Thiourea
Hydroxylamine
Fumarate
Sodium borohydride
L-2-Thiohistidine
-N,N’-Dimethyl-L-2-thiohistidine
Urocanic acid
PMSF
Glyoxal sodium bisulfite
1

Figure legends
Scheme 1. Reaction catalyzed by thiourocanate hydratase.
Figure 1. Overexpression and purification of the thiourocanate-metabolizing
enzyme.
Protein samples from various stages of the purification process were subjected
to SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1, marker
proteins (sizes in kDa are shown); lane 2, crude extract; lane 3, following
Toyopearl–DEAE 650M chromatography; lane 4, following Toyopearl–Butyl
650M chromatography; lane 5, following Toyopearl–AF–Blue 650M
chromatography.
Figure 2. Effect of pH and temperature on thiourocanate hydratase.
All assays were carried out in triplicate and all data are expressed as the mean
± standard deviation. (A) The enzyme activity was measured in 0.1 M
potassium phosphate (closed circle), 0.1 M HEPES (closed square), 10 mM
sodium pyrophosphate (open circle), and 0.1 M glycine-NaOH (closed triangle)
buffers at 30 °C. The reaction mixtures contained 80 M thiourocanic acid, 17
g/ml thiourocanate hydratase, and buffer. Although sodium acetate and
Tris-HCl inhibited the enzymatic reaction, the enzyme activity was measured in
0.1 M sodium acetate (pH 6.0–6.5) and 0.1 M Tris-HCl (pH 7.5–8.5) buffers. The
relative activities of the enzyme were 1.3 ± 0.26% (sodium acetate, pH 6.0), 3.3
± 0.73% (sodium acetate, pH 6.5), 20 ± 2.6% (Tris-HCl, pH 7.5), 32 ± 2.9%
(Tris-HCl, pH 8.0), and 43 ± 3.3% (Tris-HCl, pH 8.5). (B) The enzyme activity
was assayed at 25–75 °C. The reaction mixture contained 80 M thiourocanic
acid, 0.1 M potassium phosphate buffer (pH 7.2), and 17 g/ml thiourocanate
hydratase. (C) The enzyme solutions containing 3.4 mg/ml thiourocanate
1

hydratase, 20% glycerol, 50 mM NaCl, and 0.1 M buffers with different pH
values [sodium acetate (open triangle), potassium phosphate (closed circle),
Tris-HCl (open circle), and glycine-NaOH (closed triangle)] were incubated at
30 °C for 1 h. Subsequently, the enzyme assay was performed with a reaction
mixture containing 80 M thiourocanic acid, 0.1 M potassium phosphate buffer
(pH 7.2), and 17 g/ml incubated thiourocanate hydratase. The enzyme activity
of the untreated thiourocanate hydratase was assayed in triplicate and the
mean of the measurements was defined as 100% remaining activity. (D) The
enzyme solution containing 3.4 mg/ml thiourocanate hydratase, 20% glycerol,
50 mM NaCl, and 0.1 M potassium phosphate buffer (pH 7.2) was incubated at
4–70 °C for 1 h. Subsequently, the enzyme assay was performed with a
reaction mixture containing 80 M thiourocanic acid, 0.1 M potassium
phosphate buffer (pH 7.2), and 17 g/ml incubated thiourocanate hydratase.
The enzyme activity of the untreated thiourocanate hydratase was assayed in
triplicate and the mean of the measurements was defined as 100% remaining
activity.
Figure 3. Alignment of the amino acid sequences of urocanase from P. putida
(PpURO) (26) and thiourocanate hydratase from Burkholderia sp. HME13
(TUH).
Asterisks indicate the glycine-rich sequence fingerprint and the arrow indicates
the Glu197 residue.
Figure 4. Putative ergothioneine utilization pathway and histidine utilization
(Hut) pathway.
Histidine is metabolized to formiminoglutamic acid by three enzymatic reactions.
In some genera (e.g., Klebsiella and Bacillus), formiminoglutamic acid is
hydrolyzed to formamide and glutamic acid (Pathway 1). In other genera (e.g.,
2

Pseudomonas and Streptomyces), formiminoglutamic acid is metabolized to
glutamic acid via the formyl glutamic acid (Pathway 2).
3

Manuscript Submitted to JB
Page 30 of 34
+
H2O
Muramatsu et al. Scheme 1