570 Notes Biol. Pharm. Bull. 24(5) 570—574 (2001) Vol. 24, No.

Cloning and Sequencing of the astA Gene Encoding Arylsulfate

Sulfotransferase from Salmonella typhimurium
Jin-Wook KANG,a Ae-Ran KWON,a Dong-Hyun KIM,b and Eung-Chil CHOI*,a
College of Pharmacy, Seoul National University,a Seoul 151–742, Korea and College of Pharmacy, Kyunghee University,b
Seoul 131–701, Korea. Received November 9, 2000; accepted February 16, 2001

Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic ac-
ceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library of Salmonella
typhimurium. The gene was subcloned into the vector pKF3 and was sequenced. A recombinant clone harboring
the gene was directly identified using a fluorescent assay. Sequencing revealed two contiguous open reading
frames (ORFs) on the same strand. Based on amino acid sequence homology, ORF1 and ORF2 are designated as
astA and dsbA, respectively. The deduced amino acid sequence of astA from S. typhimurium was highly similar to
those of the Enterobacter amnigenus, Klebsiella, and Campylobacter jejuni ASSTs, encoded by the astA genes.
However, an ASST activity assay revealed a different acceptor specificity. Using p-nitrophenyl sulfate (PNS) as a
donor substrate, phenol is the best acceptor substrate, followed by a -naphthol, resorcinol, tyramine, aceta-
minophen, and tyrosine.
Key words arylsulfate sulfotransferase; astA gene; Salmonella typhimurium; molecular cloning; sequencing

Phenol sulfation is considered to be a major metabolic MATERIALS AND METHODS

pathway for the detoxification of endogenous and exogenous
compounds bearing phenolic functional groups.1) It is cat-
alyzed by phenol sulfotransferase (PST), an enzyme that has
been purified from several mammalian organs, including
liver, lung, brain, kidney, epithelial cells, and erythrocytes.2,3)
Besides the liver and extrahepatic tissues, it is possible that
intestinal flora also play an important role in the detoxifica-
tion of phenolic chemicals by sulfoconjugation.4,5) PST cat-
alyzes the transfer of the sulfate group from 39-phospho-
adenosine-59-phosphosulfate (PAPS) to an acceptor com-
pound to form a sulfate ester.6,7)
Kim et al. isolated arylsulfate sulfotransferase (ASST)
producing bacteria, Eubacterium A-44,8) Klebsiella K-36,9)
and Haemophilus K-12,10) from human, rat, and mouse in-
testinal flora, respectively. Kwon et al. also isolated the
ASST from Enterobacter amnigenus AR-37.11) These bacter-
ial ASSTs characteristically require a phenolic sulfate ester
as the donor substrate, whereas PAPS is the donor for the
mammalian tissue PSTs.12,13) The ASST protein also cat-
alyzes the sulfation of the tyrosine residues of peptides and
proteins such as kyotorphin, enkephalin, cholecystokinin-8,
trypsin inhibitor, and insulin.14) In addition, the antithrombin
activity of recombinant hirudin, which had been sulfated by
the ASST obtained from Eubacterium A-44, was increased
about 3.4-fold as compared to that of the unsulfated
hirudin.15)
In contrast to the numerous studies of tissue PSTs, very
little is known about the enzymatic sulfation by intestinal
bacteria. As a preliminary experiment to determine why this
enzyme exists, the distribution of bacteria with ASST activ-
ity was investigated, by analyzing about 1300 bacterial
strains maintained in our laboratory. In view of the results
obtained thus far, it was found that the enzyme activity was
not detected in certain bacteria, and therefore, it might not be
essential enzyme for the viability.16) Salmonella typhimurium
ATCC 13311, which has high ASST activity, was selected for
further genetic characterization. In this paper, the cloning
and sequencing of the S. typhimurium astA gene, an arylsul-
fate sulfotransferase determinant, is described.
∗ To whom correspondence should be addressed. e-mail: ecchoi@snu.ac.kr
Bacterial Strains, Phage, Plasmids, and Growth Condi-
tions S. typhimurium was grown in Luria-Bertani (LB)
broth at 37 °C for 12 h. Escherichia coli LE392 was used as
the cloning host for l GEM-11 (Promega, Madison, WI)
phage clones, and E. coli TH2 was used for subcloning and
DNA sequencing. These strains were also grown in LB broth
at 37 °C for 12 h. The plasmid pKF3 (Takara Shuzo, Tokyo,
Japan) was used as the cloning vector and the subcloning
vector for sequencing. In the case of phage, about 13106
plaque forming units (PFU) of recombinant phage were
mixed with 0.1 ml of an overnight culture of E. coli LE392,
and the mixture was incubated at 37 °C for 20 min. The mix-
ture was added to 10 ml of prewarmed LB medium (supple-
mented with 0.1 ml of 1 M MgSO4), and the flask was shaken
at 37 °C for 8 h until the medium turned cloudy. For the se-
lection of astA transformants, 0.1 mM 4-methylumbelliferyl
sulfate (4-MUS) was added to the LB medium. Streptomycin
and chloramphenicol were also added to the LB medium, at
concentrations of 50 and 12 m g/ml, respectively.17)
Molecular Cloning of astA from Salmonella typhi-
murium ATCC 13311 A S. typhimurium ATCC 13311 ge-
nomic library was constructed using l GEM-11, according to
the manufacturer’s recommendation (Promega, Madison,
WI). The chromosomal DNA from S. typhimurium ATCC
13311 was isolated and partially digested with Sau3AI. Frag-
ments ranging from 15 to 23 kb were then fractionated by
agarose gel electrophoresis, purified, and ligated between the
arms of l GEM-11 cleaved with BamHI. The ligation mix-
ture was incubated with an in vitro packaging system (Packa-
gene, Promega, Madison, WI), and the resulting phage parti-
cles were propagated on strain E. coli LE392.
We successfully detected recombinants of interest by di-
rectly screening for activity in phage plaques. The sulfate of
the 4-MUS on the medium was cleaved by the ASST activity,
and then 4-methylumbelliferone, a fluorescent product, was
produced. Positive plaques were monitored by UV fluores-
cence (320 nm). By cleaving the recombinant phage DNA
with SacI and electrophoresing them on a 0.4% (w/v)
© 2001 Pharmaceutical Society of Japan
May 2001
agarose gel, it was found that the smallest recombinant phage
DNA had a 16 kb insert.
For subcloning, Sau3AI-partially digested insert fragments
were ligated into the BamHI-digested pKF3 vector with T4
DNA ligase (Promega, Madison, WI). This ligation mixture
was used to transform competent E. coli TH2 cells. The
colonies carrying the subcloned plasmid were screened again
for enzyme activity by plating them on an LB plate contain-
ing 4-MUS as the substrate.
Nucleotide Sequencing and Sequence Analysis By
using the various subclones in pKF3, the sequence of the re-
gion containing the astA gene was determined by the dideoxy
chain termination method18) using a sequencing kit (Bioneer,
Taejon, Korea). DNA sequencing was performed with the
pKF3 primers F3 and R2 (Takara, Tokyo, Japan). Both of the
DNA strands were sequenced by a combination of specific
oligonucleotide primers on an automated DNA sequencer
(Model 4000, Li-Cor, Lincoln, NE, U.S.A.).
Homology searches with the deduced amino acid se-
quence of ASST were done using the BLAST network ser-
vice19) at NCBI and the CLUSTAL W program.20)
ASST Activity Assay The preparation of cell extracts
from S. typhimurium ATCC 13311, E. coli LE392, and E.
coli TH2 harboring the astA gene was carried out as previ-
ously described.21)
The assay mixture (total volume of 0.63 ml) contained
30 m l of 50 mM p-nitrophenyl sulfate (PNS), 0.29 ml of
20 mM phenol (and occasionally other acceptors at the same
concentration), 0.21 ml of 0.1 M Tris–HCl, pH 8.0, and 0.1 ml
of the cell extract. This assay mixture was incubated at 37 °C
for 20 min, and the reaction was stopped by the addition of
0.4 ml of 1 N NaOH. The presence of ASST was detected by
the appearance of a yellow color. Quantitative measurements
were also performed by measuring the absorbance at 405 nm.
Nucleotide Sequence Accession Number The nu-
cleotide sequence data reported in this paper will appear in
the EMBL and NCBI nucleotide sequence databases under
accession number AF295574.
RESULTS AND DISCUSSION
Cloning of astA from S. typhimurium ATCC 13311 E.
coli LE392 cells, infected with the phage library of S. ty-
phimurium ATCC 13311 chromosomal DNA in l GEM-11
were cultured on LB agar containing 4-MUS. The ASST ac-
tivity-positive plaques, which produced blue fluorescent light
under UV illumination (320 nm), were isolated and purified.
Restriction analysis revealed that the smallest phage DNA
had a 16 kb insert. This recombinant phage was designated as
l JW55.
For subcloning, Sau3AI-partially digested fragments of the
16 kb insert DNA were ligated with BamHI-digested pKF3,
and the ligation mixture was used to transform E. coli TH2.
Many positive colonies harboring recombinant plasmids
were detected by fluorescence and enzyme activity. The
smallest recombinant plasmid, harboring a 3.3 kb DNA in-
sert, was designated as pJW55 and underwent further analy-
sis.
Nucleotide Sequence Analysis The restriction map of
pJW55 and the locations of the ORFs are depicted in Fig. 1.
Various restriction enzymes were used to generate the restric-
571
Fig. 1. Partial Restriction Map of the pJW55
The solid box indicates dsbA gene, while astA is symbolized by the hatched box. The
direction of transcription of these different genes is shown by arrows. Restriction en-
zyme sites: B, BglII; Na, NaeI; E, EcoRI; S, SacII; Nr, NruI; H, HpaI; C, ClaI; A,
AvaII; X, XmnI.
tion enzyme map of the insert of pJW55. A total of 2826 bp
was sequenced and revealed the presence of two contiguous
ORFs on the same strand. Figure 2 shows the complete nu-
cleotide sequences of the two ORFs, with the corresponding
deduced amino acid sequences. ORF1 is 1794 nucleotides
long, beginning with an ATG start codon at position 222 and
ending with a TAA stop codon at position 2016. A promoter-
like sequence22) is located upstream from the start codon,
with a 235 sequence of TTATTAT followed by a 14 bp space
and a 210 region of TATATT. A potential ribosome-binding
site (TAAGGAG) is present 8 bp upstream of the predicted
ATG start codon, which is similar to that of E. coli23) (Fig. 2).
A database search using the BLAST search programs re-
vealed that the S. typhimurium ORF1 amino acid sequence
shares high similarities with the ASST proteins from Enter-
obacter11) and Klebsiella,21) and the arylsulfatase from
Campylobacter jejuni,24) and thus ORF1 was designated as
astA. The protein sequence deduced from the S. typhimurium
astA gene is similar to those from the astA genes of E. amni-
genus AR-3711) (GenBank accession no. AF012826) (87%
identity and 93% similarity), Klebsiella K-3621) (GenBank
accession no. U32616) (86% identity and 93% similarity),
and the arylsulfatase from C. jejuni24) (GenBank accession
no. U38280) (40% identity and 59% similarity) (Fig. 3). The
higher homology of the S. typhimurium enzyme with those of
E. amnigenus and Klebsiella, as compared to that of C.
jejuni, agrees well with the phylogenetic distance. The Sal-
monella, Enterobacter, and Klebsiella genera belong to
group 5 of the Enterobacteriaceae family, but the Campy-
lobacter genus belongs to group 2.25)
The alignment of the four apparent ASSTs from S. ty-
phimurium, E. amnigenus,11) Klebsiella,21) and C. jejuni 24)
shows conserved regions, which will be helpful to locate
some active sites. The active sites might contain a tyrosine
residue, which is the only amino acid containing phenolic hy-
droxy group. At present, eleven conserved tyrosine residues
have been identified, and it will be necessary to elucidate the
possible active sites by site-directed mutagenesis.
ORF2 consists of 689 bp, starting with an ATG start codon
at nucleotide 2038 and ending with a TAA stop codon at po-
sition 2707. The putative ribosome-binding site (GAAGGA)
is located 12 to 7 bp upstream of the start codon of the
ORF2. A BLAST search of GenBank revealed that the pre-
dicted protein encoded by ORF2 showed a significant match
to the dsbA genes,26) which encode an enzyme involved in
disulfide bond formation, of E. amnigenus AR-3711) (90%
identity and 96% similarity) and Klebsiella K-3621) (88%
identity and 95% similarity). The subclones containing dsbA
did not exhibit the ASST activity (data not shown). DsbA
acts to facilitate the formation of disulfide bonds of many
572 Vol. 24, No. 5

Fig. 2. Nucleotides and Deduced Amino Acid Sequences of the astA and dsbA Genes of Salmonella typhimurium ATCC 13311
The predicted 235 and 210 regions of the putative promoter and the putative ribosome binding sites (RBS) are indicated. Stop codons are marked with asterisks.
May 2001 573

Fig. 3. CLUSTAL W Multiple Alignment of ASST from Salmonella typhimurium ATCC 13311 with ASSTs from Enterobacter amnigenus AR-37, Kleb-
siella K-36, and Arylsulfatase from Campylobacter jejuni
Asterisks below the aligned sequences indicate amino acid present in all four sequences. Positions with colons below contain strongly conserved regions, and periods indicate
more weakly conserved residues. Gaps introduced to align the protein sequences optimally are indicated by dashes.

proteins in periplasmic space.26)
Substrate Specificity The acceptor substrate specificity
of the ASST protein is presented in Table 1. The phenyl sul-
fate esters are unique donor substrates for ASST, but PAPS,
the donor substrate of mammalian sulfotransferase, is inef-
fective. When PNS is used as a donor, the best acceptor sub-
strate of S. typhimurium ATCC 13311 ASST is phenol, fol-
lowed by a -naphthol, resorcinol, tyramine, acetaminophen,
and tyrosine. The present enzyme is quite different in its ac-
ceptor specificity from the enzyme purified from Eubac-
terium A-44,8) and is similar to those from E. amnigenus AR-
37,11) Klebsiella K-36,21) and Haemophilus K-12.10)
574 Vol. 24, No. 5

Table 1. Acceptor Substrate Specificity of ASST 3) Sekura R. D., Duffel M. W., Jakoby W. B., Methods Enzymol., 77,
197—206 (1981).
a)
Relative activity (%) 4) Kim D. H., Kobashi K., Biochem. Pharmacol., 35, 3507—3510
Acceptor (1986).
Salmonella typhimurium Recombinant 5) Kim D. H., Konishi L., Kobashi K., Biochim. Biophys. Acta, 872, 33—
41 (1986).
Phenol 100 100 6) Sekura R. D., Jakoby W. B., J. Biol. Chem., 254, 5658—5663 (1979).
p-Acetaminophen 4.3 4.3 7) Spencer B., Biochem. J., 77, 294—304 (1960).
Tyramine 6.5 6.9 8) Kim D. H., Konishi L., Kobashi K., Biochem. Biophys. Acta, 872,
Tyrosine 4.1 3.8 33—41 (1986).
a -Naphthol 83.8 79.3 9) Kim D. H., Kim H. S., Kobashi K., J. Biochem., 112, 456—460
Resorcinol 54.2 49.5 (1992).
10) Lee N. S., Kim B. T., Kim D. H., Kobashi K., J. Biochem., 118, 796—
a) Acceptor substrate specificity was measured by using PNS as a donor substrate. 801 (1995).
Absorbance of assay mixture (PNS1Phenol) at 405 nm was taken as 100%. 11) Kwon A. R., Oh T. G., Kim D. H., Choi E. C., Protein Expression
Purif., 17, 366—372 (1999).
The astA gene encodes a protein that possesses a unique 12) Kim D. H., Kobashi K., J. Biochem., 109, 45—48 (1991).
enzymatic activity. The astA coding sequence could be used 13) Kim D. H., Kim H. S., Imamura L., Kobashi K., Biol. Pharm. Bull.,
as a reporter gene. In this experiment, the standard spec- 17, 543—545 (1994).
14) Kobashi K., Kim D. H., Biochem. Biophys. Res. Commun., 140, 38—
trophotometric assay, which is based on the production of 42 (1986).
PNP, was used. Moreover, the ASST activity can also be 15) Muramatsu R., Nukui E., Sukesada A., Misawa S., Komatsu Y.,
measured accurately using a fluorometric assay. The sulfate Okayama T., Wada K., Morikawa T., Hayashi H., Kobashi K., Eur. J.
of the 4-MUS is cleaved by the ASST activity, and 4-methy- Biochem., 223, 243—248 (1994).
lumbelliferone, a fluorescent product, is produced. This fluo- 16) Baek M. C., Kwon A. R., Chung Y. J., Kim B. K., Choi E. C., Arch.
Pharm. Res., 21, 475—477 (1998).
rometric assay is very specific, extremely sensitive, inexpen- 17) Sambrook J., Fritsch E. F., Maniatis T., “Molecular Cloning: A Labo-
sive, and rapid. Therefore, it would be very desirable to de- ratory Manual,” 2nd ed., Cold Spring Harbor Laboratory Press, Cold
velop the astA coding region as a reporter gene system. Spring Harbor, New York, 1989.
We plan to elucidate the active sites of the proteins by site- 18) Sanger F., Nicklen S., Coulson A. R., Proc. Natl. Acad. Sci. U.S.A., 74,
5463—5467 (1977).
directed mutagenesis of the ast genes. Furthermore, we will
19) Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J., J. Mol.
clarify the attribution of Dsb family proteins for the ASST to Biol., 215, 403—410 (1990).
achieve disulfide bond formation and correct folding. 20) Thompson J. D., Higgins D. G., Gibson T. J., Nucleic Acids Res., 22,
4673—4680 (1994).
Acknowledgement This study was supported in part by 21) Baek M. C., Kim S. K., Kim D. H., Kim B. K., Choi E. C., Microbiol.
Immunol., 40, 531—537 (1996).
the 1999 and 2000 BK21 projects for Medicine, Dentistry, 22) Harley D. K., Reynolds R. P., Nucleic Acids Res., 15, 2343—2361
and Pharmacy. (1987).
23) Shine J., Dalgarno L., Proc. Natl. Acad. Sci. U.S.A., 71, 1342—1346
REFERENCES (1974).
24) Yao R., Guerry P., J. Bacteriol., 178, 3335—3338 (1996).
1) Dodgson K. S., Tudball N., Biochem. J., 74, 154—159 (1960). 25) Holt J. G., Krieg N. R., Sneath P. H. A., Staley J. T., Williams S. T.,
2) Roy A. B., “Sulfotransferases, Sulfation of Drugs and Related Com- “Bergey’s Manual of Determinative Bacteriology,” 9th ed. The
pounds,” ed. by Mulder G. J., CRC Press, Boca Raton FL., 1981, pp. Williams & Wilkins Co., Baltimore, Md., 1994.
131—185. 26) Bardwell J. C., McGovern K., Beckwith J., Cell, 67, 581—589 (1991).