Aryl sulfotransferase from Haliangium ochraceum: A versatile tool for the sulfation of small molecules

Article abstract of DOI:10.1002/cctc.201300853

Sulfation is an important molecular modification that regulates essential cellular processes and is also implicated in numerous pathological processes. The enzymes responsible for this reaction in living organisms are sulfotransferases. The gene Hoch-5094 from Haliangium ochraceum is annotated as a putative sulfotransferase. The arylsulfotransferase codified by this gene (HocAST) was expressed heterologously in E. coli and showed aryl sulfotransferase activity. Circular dichroism analysis of HocAST showed a main α/β secondary structure that agrees with the overall structure of other cytosolic sulfotransferases. Interestingly, HocAST was able to use both p-nitrophenyl sulfate and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as sulfuryl donors contrary to that of aryl sulfate sulfotransferase, which cannot use PAPS as a donor. Regarding the specificity towards the acceptor, HocAST has shown quite a wide scope and was able to accept several mono- and dihydroxylated phenols and other phosphorylated compounds as substrates. Sulfation variations: We prove experimentally that the gene Hoch-5094 from Haliangium ochraceum encodes for an aryl sulfotransferase. The codified enzyme, HocAST, may be a very versatile biocatalyst as it is able to use both p- nitrophenyl sulfate (p-NPS) and 3'-phosphoadenosine-5′-phosphosulfate (PAPS) as donors and transfer the sulfuryl group to several phenolic compounds and biologically relevant phosphorylated molecules. GTP(S)=Guanosine-5′- triphosphate(-5′-sulfate), BiPhOH= 4,4'-Biphenol, BiPhOS=4,4'-Biphenol 4-sulfate.

Full text of DOI:10.1002/cctc.201300853

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DOI: 10.1002/cctc.201300853
Aryl Sulfotransferase from Haliangium ochraceum: A
Versatile Tool for the Sulfation of Small Molecules
Ivꢀn Ayuso-Fernꢀndez, Miquel A. Galmꢁs, Agatha Bastida, and Eduardo Garcꢂa-Junceda*
[
a]
Sulfation is an important molecular modification that regulates
essential cellular processes and is also implicated in numerous
pathological processes. The enzymes responsible for this reac-
tion in living organisms are sulfotransferases. The gene Hoch_
with the overall structure of other cytosolic sulfotransferases.
Interestingly, HocAST was able to use both p-nitrophenyl sul-
fate and 3’-phosphoadenosine-5’-phosphosulfate (PAPS) as sul-
furyl donors contrary to that of aryl sulfate sulfotransferase,
which cannot use PAPS as a donor. Regarding the specificity
towards the acceptor, HocAST has shown quite a wide scope
and was able to accept several mono- and dihydroxylated phe-
nols and other phosphorylated compounds as substrates.
5
094 from Haliangium ochraceum is annotated as a putative
sulfotransferase. The arylsulfotransferase codified by this gene
HocAST) was expressed heterologously in E. coli and showed
(
aryl sulfotransferase activity. Circular dichroism analysis of
HocAST showed a main a/b secondary structure that agrees
Introduction
Sulfation is an important molecular modification in all living or-
ganisms that regulates essential cellular processes and is also
complex polysaccharides such as glycosaminoglycans (GAGs),
which are polymers that play crucial roles in the development
and organogenesis and thus seem to be essential for multicel-
[
1]
implicated in numerous pathological processes. The enzymes
responsible for this reaction in living organisms are sulfotrans-
[4]
lular life. However, the synthetic use of STs is hampered be-
cause of the high cost and instability of PAPS and the problem
of product inhibition caused by 3’-phosphoadenosine-5’-phos-
ferases (EC 2.8.2.-). These enzymes catalyze the transfer of a sul-
À
furyl moiety (SO ) from 3’-phosphoadenosine-5’-phosphosul-
3
[5]
fate (PAPS) to the hydroxyl and primary amine groups of a vari-
ety of acceptors. In eukaryotes, two classes of sulfotransferases
have been described: Golgi-membrane sulfotransferases (STs),
which sulfate large molecules such as polysaccharides and pro-
teins, and cytosolic sulfotransferases (SULTs), which transfer the
sulfuryl group from PAPS to phenols, steroids, hormones,
amines, and xenobiotics. This latter family is also known as
phate (PAP). In addition to their use for the sulfation of differ-
[6]
ent flavonoids, steroids, peptides, and aliphatic alcohols,
ArylST, in particular rat liver aryl sulfotransferase IV (Ast IV), has
been used to develop a one-enzyme regeneration system for
[7]
PAPS (Scheme 1). This cycle is based on the reversibility of
[
2]
phenol or aryl sulfotransferases (ArylST). In addition to these
two families, a group of PAPS-independent sulfotransferases
that use two aryl substrates, one as a sulfuryl donor and the
other one as an acceptor, have been described in bacteria. Al-
though the bacterial aryl sulfate sulfotransferase (ASST) was
first described in commensal intestinal bacteria as early as
1
986, the identity of the natural donor has not been clearly es-
[
3]
tablished.
From a synthetic point of view, STs are attractive because of
their ability to sulfate, in a regio- and stereoselective manner,
+
[
[
a] I. Ayuso-Fernꢀndez, M. A. Galmꢁs, Dr. A. Bastida, Dr. E. Garcꢂa-Junceda
Departamento de Quꢂmica Orgꢀnica Biolꢃgica
Instituto de Quꢂmica Orgꢀnica General, CSIC
Juan de la Cierva 3, 28006 Madrid (Spain)
Fax: (+34)915-644-853
Scheme 1. PAPS regeneration cycle based on the use of aryl sulfo-
transferases.
E-mail: eduardo.junceda@csic.es
+
[8]
] Current address:
the reaction catalyzed by the ArylST. Thus, if coupled to an-
Departamento de Biologꢂa Medioambiental
Centro de Investigaciones Biolꢃgicas, CSIC
Ramiro de Maeztu 9, 28040 Madrid (Spain)
other PAPS-dependent sulfotransferase, the ArylST can transfer
the sulfuryl group from p-nitrophenyl sulfate (p-NPS) to PAP to
regenerate PAPS in situ and overcome the inhibition by PAP.
However, scale-up of this regeneration system is hampered by
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201300853.
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[
6c]
the poor stability of the pure Ast IV. Therefore, the search for
a new ArylST with better properties as a biocatalyst is ongoing.
In this sense, the gene Hoch_5094 from the bacteria Halian-
gium ochraceum codifies for a putative aryl sulfotransferase.
Herein, we describe the heterologous expression of the gene
Hoch_5094 in Escherichia coli and the biochemical characteriza-
tion of the gene product as well as a preliminary analysis of its
donor and acceptor specificity.
A: This domain is known as the phosphosulfurile binding
site (PBS) and interacts with the 5’-phosphate group from
PAPS to point the sulfuryl donor to the acceptor in the active
site.
B: This highly conserved region includes the catalytic histi-
dine (H105 in HocAST). It is expected that the amino acids in
this domain contribute significantly to the structure of the
active center.
C: In HsSULT1A1a R130 and S138 interact with the 3’-phos-
phate group of PAPS. In addition, S138 avoids the hydrolysis of
PAPS in the absence of an acceptor substrate.
Results and Discussion
D: This domain contains the motif GxxGxWK, which is
highly conserved in this kind of sulfotransferase. This sequence
interacts with the 3’-phosphate group of PAPS and, together
with domain C, stabilizes its binding to the active site. In
HocAST there is an R instead of the K but as both amino acids
are basic and have similar properties, it can be expected that
the role of this domain is conserved in this enzyme.
We performed automated protein structure homology mod-
Sequence analysis of the aryl sulfotransferase from
Haliangium ochraceum
In recent years the number of complete genomes, especially
from bacteria that have been sequenced, has grown dramati-
cally, which provides a unique source to find new enzymes.
Haliangium ochraceum is the type species of the genus Halian-
gium in the myxococcal family Haliangiaceae and its genome
[
12]
[
9]
eling by using the SwissModel
server using the protein
has been sequenced recently. The product of the gene
Hoch_5094 is annotated as a putative sulfotransferase because
of the sequence homology with the family SULT_
[13]
SULT1D1 from Mus musculus (PDB entry 2zyt) as a template.
The folding of HocAST is highly conserved if superposed to
other sulfotransferases, the structures of which have been
solved already. The topology of HocAST folding was b1, a1,
a2, a3, b2, b3, a4, b4, b5, a5, a6, b6, a7, a8, a9, a10, a11,
a12, which is virtually identical to the topology of the SULT
proteins (Figure 2A). To confirm that the catalytic residue of
the HocAST could be His105 (deduced by alignment), we per-
formed docking studies of the model protein with
[
10]
1
(Pfam00685). As there is no experimental evidence of the
activity of this enzyme, we decided to undertake a comparative
analysis of its sequence with the well-characterized SULT from
human (HsSULT1A1a), rat (RnSULT1A1), and mouse
(
MmSULT1D1). The domains that define the phenol sulfotrans-
[
11]
ferase activity are shown in Figure 1.
PAP and p-NPS and obtained a glide score of
À1
[14]
À8.59 kcalmol (Figure 2B).
In the HocAST–PAP
complex, the 5’-phosphate group of PAP is posi-
tioned in an analogous manner to the phosphate
group of PAPS in the crystallographic MmSULT1D1–
PAPS complex (Figure S1). In both cases, the phos-
phate groups are 3 ꢄ from the catalytic His residue
(
His105in HocAST and His108in MmSULT1D1).
Therefore, as HocAST presented the main structur-
al characteristics and an overall 3D structure in agree-
ment with the cytosolic sulfotransferases, we decided
to perform its expression in E. coli to perform a de-
tailed structural and functional analysis as well as to
explore its synthetic applicability.
Expression and structural characterization of
HocAST
The gene Hoch_5094 was amplified by polymerase
chain reaction (PCR) from the genomic DNA of Hal-
iangium ochraceum using primers designed to com-
plement specifically 25 base pairs (bp) at the 5’ end
of the codifying and complementary DNA strains.
The recognition sequence for the restriction enzymes
NdeI and XhoI were introduced into the amplification
product during PCR. The resulting amplified band
had the expected size (954 bp) and was digested and
introduced into the expression vector pET-28b(+),
Figure 1. Alignment of the amino acid sequence of the hypothetical aryl sulfotransferase
from Haliangium ochraceum versus three known sulfotransferases. The boxes A, B, C, and
D frame the conserved regions involved in the aryl sulfotransferase activity.
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Figure 2. A) Modeling the tertiary structure of the protein HocAST; the reac-
tive His105 is shown in sticks and the conserved motifs in color: Box A, den-
sity blue; Box B, cyan; Box C, deep blue, and Box D, light blue; B) Docking of
p-NPS and PAP in the HocAST model.
which introduces a six-histidine tag at the N terminus of the
recombinant protein.
E. coli BL21 (DE3) cells were transformed with this plasmid,
and the expression of recombinant protein was induced with
0.5 mm isopropyl b-d-1-thiogalactopyranoside (IPTG) at an op-
tical density at 600 nm (OD600nm) of 0.5–0.6. Sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of
HocAST expression showed a band of the expected molecular
mass (37 kDa) in the soluble fraction that represented 70% of
the total protein. HocAST was purified by immobilized metal-
affinity chromatography (IMAC) through the His-tag.
The purified recombinant protein presented a mass of
37221 Da, and the peptide mass fingerprint showed 18 pep-
tides that cover the sequence of HocAST and identifies it un-
equivocally (Figure 3). The quaternary structure of the enzyme
was analyzed by size-exclusion chromatography. The recombi-
Figure 4. Spectroscopic characterization of recombinant HocAST. a) Far-UV
CD spectrum. b) Tryptophan fluorescence emission spectrum; FI=fluores-
cence intensity.
The tertiary structure of the protein in the environment of
the fluorophores was analyzed by fluorescence emission spec-
troscopy of the 12 tryptophan and 12 tyrosine residues con-
tained in the sequence of HocAST (Figure 4b). Upon excitation
at 275 nm, the spectrum exhibited an emission maximum cen-
tered at 335 nm, which is shifted approximately 15 nm from
the position of the maximum described for free tryptophan in
Figure 3. Peptide mass fingerprint of HocAST. The sequence of the identified
peptides is shaded and underlined. The molecular mass of each peptide is
in [Da].
[15]
aqueous solution (348–350 nm). This indicates that the aver-
age microenvironment of tryptophan in HocAST is significantly
more hydrophobic than that of a residue completely exposed
to the solvent. This blueshift of the fluorescence, together with
the CD spectrum, supports the idea that the protein is ex-
pressed with proper folding. Moreover, emission spectra ob-
tained upon excitation at 275 and 295 nm are practically iden-
tical, which indicates an almost negligible contribution of tyro-
sine residues to the fluorescence spectrum of the protein. This
contribution, lower than expected for a Trp/Tyr ratio of 12:12,
is probably because of an extensive resonance energy transfer
from tyrosine fluorescence to nearby tryptophan residues and/
or to quenching by other close side chains.
nant protein in the chromatographic column was eluted in
two peaks at 100 and 150 mL (Figure S2). Peak 1 corresponds
to the exclusion volume that contains oligomers of the protein.
If the elution volume of peak 2 was interpolated into the cali-
bration curve, a weight of approximately 40 kDa that corre-
sponds to the monomer of HocAST was obtained (Figure S2).
The spectroscopic characterization of purified HocAST is
shown in Figure 4. The far-UV circular dichroism (CD) spectrum
shows two minima in ellipticity at approximately 210 and
220 nm, indicative of a main a/b secondary structure (Fig-
ure 4a).
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Biochemical characterization of HocAST
The melting temperature of the recombinant HocAST was
determined by thermal shift experiments at 338C (see Experi-
mental Section and Figure S5). We observed a significant de-
crease of the half-life of the enzyme between 25 and 378C
(Figure 5). At 258C the half-life of the sulfotransferase was over
3 h, but at 378C it was only 36–37 min, and at 458C less than
6 min (Figure 5). However, at 48C the enzyme retained over
90% of its activity for several days.
The course of the reaction catalyzed by the recombinant
HocAST was followed in the sense of PAPS formation by moni-
toring the increase in absorbance at 405 nm caused by the ap-
pearance of para-nitrophenol(p-NP; 1). The increase in absorb-
ance was linear with enzyme concentration and reaction time.
These results indicate clearly that HocAST was able to transfer
the sulfuryl group from p-NPS to PAP (2) to show aryl sulfo-
transferase activity. The initial rate of the reaction measured
over a range of substrate concentrations showed a Michaelis–
Menten behavior (Figure S3). The apparent kinetics parameters
of the HocAST enzyme were calculated independently for both
substrates p-NPS and PAP (Table 1). It is difficult to compare
Substrate specificity studies
For this preliminary study of the HocAST substrate specificity,
we assayed different commercially available phenolic com-
pounds, aliphatic alcohols, and biologically relevant phos-
phorylated compounds that can be considered PAP analogs
(
Scheme 2).
[
a]
Table 1. Apparent kinetics parameters of HocAST for p-NPS and PAP.
Preliminary screening was performed by using a thermal
[17]
shift assay. The assay involves monitoring changes in the
fluorescence signal of Sypro orange dye as it interacts with the
hydrophobic core of a protein that is undergoing thermal un-
À1
À1
max
V K
MÀ1
Substrate
K
M
[mM]
V
max [mmolmin mg
]
p-NPS
PAP
45.9Æ2.2
4.0Æ0.9
0.84Æ0.008
0.76Æ0.015
0.018
0.19
folding. The melting temperature (T ) of the protein will in-
m
[a] Kinetic parameters calculated at 258C and pH 7.0.
crease in the presence of ligands that bind to the properly
folded protein. If an increase in Tm was detected, we per-
formed the sulfation reaction as described in the Experimental
Section, and the formation of the reaction product was con-
firmed by ESI-MS. The results are summarized in Table 2.
these data with others reported in the literature as they are ap-
parent parameters that are dependent on the conditions
under which the assay was performed. Nevertheless, they are
of the same order of magnitude as those reported for the sul-
fotransferase STF9 from Mycobacterium avium, another sulfo-
transferase that is also able to use PAPS and p-NPS as sulfuryl
Table 2. Screening of HocAST acceptor specificity.
[
a]
Compound
DT
m
[8C]
Activity [%]
[
16]
group donors.
3
4
5
6
7
1
1
1
2.0
0.8
–
12.0
18.0
n.a.
The recombinant HocAST exhibited activity between pH 6.0
and 7.5 with the maximum activity at pH 6.5. At pH values
over 7.5, the activity drops drastically to virtually zero (Fig-
ure S4). To study the thermal stability of HocAST, the enzyme
was incubated at 4, 25, 37, and 458C and their remnant activi-
ties were evaluated at room temperature (Figure 5). Therefore,
we determined in this assay the progressive loss of activity
caused by the irreversible denaturation of the enzyme.
[b]
[c]
–
–
–
2.0
–
n.a.
n.a.
n.a.
93.0
n.a.
n.a.
4.4
17.0
4.4
9.7
0
1
2
13
14
5
6
7
8
9
–
1.4
2.8
1.0
2.4
–
1
1
1
1
1
n.a.
n.a.
–
[
a] 100% Activity is the maximum reaction rate with PAP as acceptor and
m
p-NPS as sulfuryl donor. [b] –: No increase in T detected. [c] n.a.: Not
applicable.
HocAST is able to transfer the sulfuryl group from p-NPS to
the hydroxyl group of phenolic compounds with relatively
good activity but aliphatic alcohols were not substrates of the
enzyme. The best substrate was 4,4’-biphenol (11) probably
because of the symmetry of the molecule. In the case of cate-
chol (4), we could detect not only the monosulfated product,
+
+
but also the disulfated one ([M+2 ] 191.2 and [M+1 ] 269.8,
respectively). The assayed nucleoside triphosphates were good
acceptors for the enzyme, and the results obtained indicate
Figure 5. Thermal stability of the recombinant HocAST at 4 (*), 25 (!),
3
7 (
), and 458C (^).
&
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HocAST has shown a broad scope and it is able to
accept different phenolic compounds and nucleoside
triphosphate as substrates. Therefore, HocAST is an
interesting addition to the biocatalyst toolbox for the
enzymatic sulfation of biologically relevant
compounds.
Experimental Section
Materials and general procedures
E. coli BL21(DE3) competent cells were purchased from
Stratagene Co. (US). Restriction enzymes, Taq polymerase
and T4-DNA ligase were purchased from MBI Fermentas
AB (Lithuania). PCR primers were purchased from Isogen
Life Science (Spain), and the pET-28b(+) expression
vector was purchased from Novagen. IPTG was pur-
chased from Applichem GmBH (Germany). Plasmids and
PCR purification kits were from Promega (US), and the
DNA purification kit from agarose gels was from Eppen-
dorf (Germany). SDS-PAGE was performed by using 10
and 5% acrylamide in the resolving and stacking gels, re-
spectively. Gels were stained with Coomassie brilliant
blue R-250 (Applichem GmBH, Germany). Electrophoresis
was always run under reducing conditions in the pres-
ence of 5% b-mercaptoethanol. Nickel iminodiacetic acid
2
+
(
Ni -IDA) agarose was supplied by Agarose Bead Tech-
nologies (Spain). All other compounds were purchased
from Sigma–Aldrich and were used directly. Solvents
were of analytical grade. DNA manipulation was per-
[18]
formed according to standard procedures.
Expression and purification of the aryl sulfotrans-
ferase from Haliangium ochraceum
Hoch_5094 gene was amplified by PCR by using an
iCycler Thermal Cycler (MiniOpticon, BioRad) using ge-
nomic DNA from Haliangium ochraceum obtained from
DSMZ (German Collection of Microorganisms and Cell
cultures, DSM n8 14365) as a template. The forward
primer (NdeI restriction site underlined) was
Scheme 2. Structure of the compounds assayed in this study as possible substrates of
HocAST.
that the nucleoside moiety is needed to be recognized by the
enzyme as triphosphate was neither ligand nor substrate.
Finally, we assayed the capacity of HocAST to transfer the
sulfuryl group from PAPS to three different nitrophenols (1, 8,
and 9). The activity with 1 was approximately half of that
shown with p-NPS and PAP. With o-NP (8), the activity was
quite similar but strongly decreased with dinitrophenol 9,
which showed a residual activity (ꢀ2% of the activity shown
with p-NPS).
5’-TATAACATATGAATTCTACTGACGAACAACACA-3’
and the reverse primer (XhoI restriction site underlined) was
5
’-CTCGAGTCAGTCCGGCAGCTCGCCG-3’.
PCR amplification was performed in 5 mL of reaction mixture that
contained 20% DMSO and was subjected to 29 cycles of amplifica-
tion. The cycle conditions were set as follows: denaturation at
9
7
48C for 1 min, annealing at 508C for 2 min, and elongation at
28C for 2 min. After digestion, the purified PCR product was ligat-
ed in the pET-28b(+) vector double-digested with the same
Conclusions
restriction enzymes.
We have shown experimentally that the gene Hoch_5094 from
Haliangium ochraceum encodes for an aryl sulfotransferase. Cir-
cular dichroism analysis of the codified enzyme, HocAST,
showed a main a/b secondary structure that agrees with the
overall structure of other cytosolic sulfotransferases. HocAST is
a very versatile enzyme as it is able to use both p-nitrophenyl
sulfate and 3’-phosphoadenosine-5’-phosphosulfate as sulfuryl
donors. With regard to the specificity towards the acceptor,
For the expression of the Hoch_5094 gene, transformed E. coli
BL21 (DE3) cells, on the pET-AST6xHis plasmid, were grown in ly-
sogeny broth (LB) medium supplemented with kanamycin
À1
(
26 mgmL ) at 378C. When the culture reached an OD of 0.5–
6
00
0
.6, protein expression was induced with IPTG (0.4 mm), and the
temperature was lowered to 308C. After 18 h, cells were harvested
by centrifugation (3800ꢅg) for 30 min at 48C. Cell pellets were re-
suspended in monosodium phosphate buffer (25 mm, pH 7.4) and
[19]
treated with lysozyme and DNase for protein extraction. The so-
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lution was centrifuged for 40 min (8000ꢅg, 48C) and collected for
ty assays were performed at RT by following the increase of the
À1
À1
purification. The supernatant, which contained the recombinant
absorbance at 405 nm (ep-NP =18000m cm ) for 15 min. In a total
volume of 250 mL of reaction mixture, HEPES buffer (50 mm,
pH 7.0), p-NPS (between 0 and 0.5 mm as indicated), and acceptor
(between 0–0.02 mm as indicated) were added. The reactions were
initiated by the addition of HocAST (50 mL, 14 mm). In all cases, the
reaction was stopped by the addition of NaOH (50 mL, 1n). For the
determination of apparent kinetic constants (with the variation of
+
2
protein, was loaded onto a Ni -IDA-agarose column pre-equili-
brated with sodium phosphate buffer (20 mm, pH 7.5). The re-
combinant ArylST was eluted with the same buffer that contained
imidazole (500 mm). All the fractions that contained protein were
pooled together and dialyzed to remove the imidazole. SDS-PAGE
showed a single band that matched the expected molecular
weight of the recombinant ArylST (37 kDa).
only one substrate), initial velocities (V ) were fitted to the Michae-
0
lis–Menten equation. Kinetics parameters were calculated by using
the built-in nonlinear regression tools in SigmaPlot 11.0.
Sequence analysis and 3D modeling of HocAST
To analyze the sequence of HocAST, an alignment of four sequen-
Substrate specificity studies
[20]
ces was performed by using ClustalX. The modeling of HocAST-
was performed by using the SwissModel server with the best
template option.
Thermal shift assays were performed by using an iQ5 Real Time De-
tection System (BioRad, CA). To monitor the protein unfolding, the
fluorescent dye Sypro orange was used. The fluorescence signal of
the dye is quenched in the aqueous environment of a properly
folded protein in solution, but as the protein unfolds, it exposed
its hydrophobic residues to the environment. The dye then binds
to the hydrophobic regions and becomes unquenched. Acceptor
solutions (5 mL, 10 mm) were added to Tris buffer (45 mL, 10 mm,
pH 7.0) that contained Sypro Orange (0.5 mL in 0.5 mL) and protein
Protein analysis
Peptide mass fingerprint analysis from the SDS-PAGE band that
corresponded to the hypothetical HocAST was performed at the
Proteomic Unit of the Spanish National Center of Biotechnology
(
CNB-CSIC). Samples were digested with sequencing-grade trypsin
(
0
4.4 mm). The plates were heated from 25 to 708C at a rate of
.28Cmin . The fluorescence intensity was measured with excita-
O/N at 378C. Analysis by MALDI-TOF-MS produced peptide mass
fingerprints, and the peptides observed can be collated and repre-
sented as a list of monoisotopic molecular weights. Data were col-
lected in the m/z range of 800–3600.
À1
tion and emission wavelengths of 490 and 530 nm, respectively.
If a thermal shift was detected, sulfotransferase activity was con-
firmed by the colorimetric method described above. However, to
determinate the activity of the HocAST using PAPS as the sulfuryl
donor, the reaction mixtures contained phosphate buffer (184 mL,
The quaternary structure of HocAST was determined by size-exclu-
sion chromatography under nondenaturing conditions. The protein
was loaded into a HiLoad 26/60 SuperdexTM 75 PG column con-
trolled by the AKTA-FPLC system (GE-Healthcare Life Science). The
column was developed in phosphate buffer (50 mm, pH 7.2) that
5
0 mm, pH 7.0), PAPS (2.7 mL, 9 mm), and acceptor (7.5 mL, 1 mm).
The reactions were initiated by the addition of HocAST (50 mL,
4 mm).
1
À1
contained NaCl (0.15m) at a constant flow rate of 1.0 mLmin . A
calibration curve was plotted with the retention times of well-
known proteins with molecular weights between 15 and 70 kDa.
In all cases, the identity of the reaction products was confirmed by
+
positive-ion ESI-MS spectrometry: m/z: PAPS 506.9 [M ]; adeno-
+
sine-5’-triphosfate 5’-sulfate (ATPS) 587 [M ]; guanosine-5’-triphos-
+
phate-5’-sulfate (GTPS) 603 [M ]; citidine-5’-triphosphate-5’-sulfate
+
CD and fluorescence spectroscopy
(CTPS) 563.9 [M ]; uridina-5’-triphosphate-5’-sulfate (UTPS) 564.7
+
+
+
[
M ]; phenol sulfate 174.2 [M ]; 4,4’-biphenol sulfate 266.0 [M ];
Far-UV CD spectra were recorded in the wavelength range of 195–
2
+
+
catechol monosulfate 191.2 [M ]; catechol disulfate 269.8 [M ]; p-
nitrophenol sulfate 245.0 [NaM ]; 2,4-dinitrophenol sulfate 280.6
NaM ]
2
50 nm by using a Jasco J-815 spectropolarimeter equipped with
+
a constant-temperature cell holder Jasco PTC423-S Peltier. The pro-
tein concentration was 14 mm. The optical path length was 0.1 cm.
The contribution of the buffer was always subtracted. For each
+
[
sample, four spectra were accumulated at a scan speed of
Molecular docking studies
À1
2
0 nmmin with a bandwidth of 0.2 nm and averaged automati-
cally. The mean residue ellipticity (V) is given in units of
The crystal structure of the complex between human sulfotransfer-
ase SULT1A1 and PAP and of the mutant D249G with PAP and p-
NP were obtained from the PDB (entries 3u3j and 3u3r, respective-
ly). The receptor was prepared by using the Wizard tool of the
Schrçdinger suite. The initial conformations of PAP and p-NP were
taken from the crystal structures. Subsequently, they were pre-
pared by using LigPrep by modification of the torsions of the li-
gands and assignment of its appropriate protonation states. By
using Glide, 32 stereochemical structures were generated per com-
pound with possible states at target pH 7.0Æ2.0 by using Ionizer,
tautomerized, desalted, and optimized by the production of a low-
energy 3D structure for the ligand under the OPLS 2005 force
2
À1
À1
8
cm dmol . A value of 110 gmol was used as the mean weight
of the residue.
Tryptophan fluorescence emission spectra of HocAST were record-
ed by using a Jasco FP-8300 spectrofluorometer, and data were
treated with the SpectraManager program. The contribution of the
buffer was always subtracted. A quartz cell with a 1 cm path
length in both the excitation (275 and 295 nm) and emission (295
to 400 nm) directions was used in all the experiments. The HocAST
protein concentration was 10 mm in phosphate buffer (5.0 mm,
pH 7.5), and experiments were performed at 258C.
[21]
field with retention of the specified chiralities of the input Maes-
tro file. Then, a receptor grid was calculated for the prepared re-
ceptor such that various ligand poses bind within the predicted
site during docking. By using Glide, grids were generated with the
retention of the default parameters of a van der Waals scaling
Enzyme activity assays and steady-state kinetics analysis
The HocAST activity measurement was performed by using a high-
throughput colorimetric 96-well plate assay. Sulfotransferase activi-
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemCatChem 2014, 6, 1059 – 1065 1064

CHEMCATCHEM
FULL PAPERS
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factor of 1.00 and a charge cutoff 0.25 subjected to the OPLS 2001
force field. A cubic box of specific dimensions (15 ꢄꢅ15 ꢄꢅ15 ꢄ)
centered on the donor or acceptor was generated for the protein.
After that, SP flexible ligand docking was performed by using Glide
of Schrçdinger-Maestro v9.2.7. Final scoring was performed on
energy-minimized poses and displayed as a Glide score. The best-
docked pose was recorded for the ligand.
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We thank the Spanish Ministerio de Ciencia e Innovaciꢃn (Grants
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(Grant S2009/PPQ-1752), and the MAPFRE Foundation for finan-
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