The thioesterase Bhp is involved in the formation of β-hydroxytyrosine during balhimycin biosynthesis in amycolatopsis balhimycina

Article abstract of DOI:10.1002/cbic.200900600

The putative hydrolase gene bhp from the balhimycin biosynthetic gene cluster has been cloned and overexpressed in Escherichia coli. The corresponding enzyme Bhp was purified to homogeneity by nickel-chelating chromatography and characterized. Although Bhp has sequence similarities to hydrolases with haloperoxidase /perhydrolase activity, it did not show any enzymatic activity with standard haloperoxidase /perhydrolase substrates (e.g., monochlorodimedone and phenol red), nonspecific esterase substrates (such as p-nitrophenyl acetate, p-nitrophenyl phosphate and S-thiophenyl acetate) or the model lactonase substrate dihydrocoumarin. However, Bhp could be shown to catalyse the hydrolysis of S-β-hydroxytyrosyl-N-acetyl cysteamine thioester (β-OH-Tyr-SNAC) with 15 times the efficiency of S-L-tyrosyl-N-acetyl cysteamine thioester (L-Tyr-SNAC). This is in agreement with the suggestion that Bhp is involved in balhimycin biosynthesis, during which it was supposed to catalyse the hydrolysis of β-OH-Tyr-S-PCP (PCP= peptidyl carrier protein) to free β-hydroxytyrosine (β-OH-Tyr) and strongly suggests that Bhp is a thioesterase with high substrate specificity for PCP-bound β-OH-Tyr and not a haloperoxidase / perhydrolase or nonspecific esterase.

Full text of DOI:10.1002/cbic.200900600

DOI: 10.1002/cbic.200900600
The Thioesterase Bhp is Involved in the Formation of
b-Hydroxytyrosine during Balhimycin Biosynthesis in
Amycolatopsis balhimycina
Sri Mulyani,[a, b] Ellen Egel,[a] Claudia Kittel,[c] Suada Turkanovic,[d] Wolfgang Wohlleben,[c]
Roderich D. Sꢀssmuth,[d] and Karl-Heinz van Pꢁe*[a]
The putative hydrolase gene bhp from the balhimycin biosyn-
thetic gene cluster has been cloned and overexpressed in Es-
cherichia coli. The corresponding enzyme Bhp was purified to
homogeneity by nickel-chelating chromatography and charac-
terized. Although Bhp has sequence similarities to hydrolases
with “haloperoxidase”/perhydrolase activity, it did not show
any enzymatic activity with standard “haloperoxidase”/per-
hydrolase substrates (e.g., monochlorodimedone and phenol
red), nonspecific esterase substrates (such as p-nitrophenyl
acetate, p-nitrophenyl phosphate and S-thiophenyl acetate) or
the model lactonase substrate dihydrocoumarin. However, Bhp
could be shown to catalyse the hydrolysis of S-b-hydroxytyro-
syl-N-acetyl cysteamine thioester (b-OH-Tyr-SNAC) with 15
times the efficiency of S-l-tyrosyl-N-acetyl cysteamine thioester
(l-Tyr-SNAC). This is in agreement with the suggestion that
Bhp is involved in balhimycin biosynthesis, during which it was
supposed to catalyse the hydrolysis of b-OH-Tyr-S-PCP (PCP=
peptidyl carrier protein) to free b-hydroxytyrosine (b-OH-Tyr)
and strongly suggests that Bhp is a thioesterase with high sub-
strate specificity for PCP-bound b-OH-Tyr and not a “haloperox-
idase”/perhydrolase or nonspecific esterase.
Introduction
Balhimycin is a glycopeptide antibiotic belonging to the van-
comycin group. Balhimycin was isolated by Nadkarni et al.
from Amycolatopsis balhimycina (formerly Amycolatopsis medi-
(bhp) and one gene for a flavin-dependent halogenase
(bhaA).[3] One previous hypothesis was that each of the two
enzymes was correlated to the attachment of one chlorine to
the aglycone of balhimycin and the structurally related chloro-
eremomycin, where a similar situation was found.[4] However,
shortly thereafter it was realised that these non-haem, metal-
free “haloperoxidases” were actually perhydrolases and that
they were not involved in the halogenation step during halo-
metabolite biosynthesis.[5–6] Later it was shown by Puk et al.
that an in-frame deletion mutant in the bhp gene did not pro-
duce balhimycin anymore. However, balhimycin production
could be restored by the addition of exogenous b-hydroxytyro-
sine. A corresponding in-frame deletion mutant in the bhaB
gene, which showed significant homology to flavin-dependent
halogenases, only produced nonhalogenated balhimycin deriv-
[a] Dr. S. Mulyani, E. Egel, Prof. K.-H. van Pꢀe
Biochemistry, TU Dresden
01062 Dresden (Germany)
Fax: (+49)351-463-35506
E-mail: karl-heinz.vanpee@chemie.tu-dresden.de
terranei).[1–2] The chemical structure of the heptapeptide back-
bone of balhimycin is identical to that of vancomycin, and the
structures differ only in their glycosylation patterns. Both balhi-
mycin and vancomycin are active against methicillin-resistant
Staphylococcus aureus (MRSA) strains. Especially with Clostridi-
um strains, balhimycin shows superior bactericidal activity to
vancomycin.[1]
[b] Dr. S. Mulyani
Prodi P. Kimia, FKIP, Universitas Sebelas Maret
Jl. Ir. Sutami 36 A, Surakarta 57126 (Indonesia)
[c] Dr. C. Kittel, Prof. W. Wohlleben
Lehrstuhl fꢁr Mikrobiologie/Biotechnologie
Eberhard-Karls-Universitꢂt Tꢁbingen
Auf der Morgenstelle 28, 72076 Tꢁbingen (Germany)
In 1999, Pelzer et al. cloned and sequenced the balhimycin
biosynthetic gene cluster.[3] Analysis of this gene cluster sug-
gested that it contains two genes coding for halogenating en-
zymes, one gene for a non-haem, metal-free “haloperoxidase”
[d] S. Turkanovic, Prof. R. D. Sꢁssmuth
Institut fꢁr Chemie/FG Organische Chemie
Technische Universitꢂt Berlin, TC Gebꢂude TC2
Strasse des 17. Juni 124, 10623 Berlin (Germany)
266
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b-OH-Tyr-PCP Hydrolase
atives. Thus it was concluded that BhaB was responsible for
the incorporation of both chlorine atoms into balhimycin,
whereas Bhp was involved in the biosynthesis of the nonpro-
teinogenic amino acid b-hydroxytyrosine. Two residues of this
amino acid are present in balhimycin, vancomycin and chloro-
eremomycin as monochlorinated derivatives.[7]
(ORF18) from chloroeremomycin biosynthesis is a thioesterase
catalysing the release of b-OH-Tyr from the peptidyl carrier
protein CepK (ORF19); however, this hypothesis has also never
been verified by any in vitro experiment.
Interestingly, there are also similarities to a thioesterase from
nikkomycin biosynthesis (NikP2), in which a b-OH-His residue is
incorporated; this indicates that Bhp is involved in balhimycin
biosynthesis in a similar way to NikP2 in nikkomycin biosynthe-
sis.[13] On the other hand, the aminocoumarin antibiotics novo-
biocin, clorobiocin and simocyclinone contain b-OH-Tyr resi-
dues, even though there is no Bhp analogue involved in these
biosynthetic pathways.[14–16] This suggests that, in these biosyn-
thetic pathways, b-OH-Tyr does not exist in free form, but only
in a peptidyl-carrier-bound form.
The work presented here focuses on the investigation of the
role of the putative hydrolase Bhp in balhimycin biosynthesis.
For this purpose, we used heterologous expression and charac-
terisation of the protein with a synthetic substrate mimicking
the suggested PCP-bound natural substrate of Bhp. Here we
report that the putative hydrolase Bhp is indeed a thioesterase
responsible for b-OH-Tyr release during balhimycin biosynthe-
sis.
Heterologous expression of bhp in E. coli
Results and Discussion
Expression of bhp in E. coli BL21(DE3)pLysS by using pRSET B
as the vector resulted in insoluble protein when the strain was
grown in Hefe–Nꢃhr bouillon (HNB) medium at 378C, with in-
duction by isopropyl-b-thiogalactopyranoside (IPTG; 4 mm) for
16 h. However, growing the strain at 308C in a medium sup-
plemented with sorbitol (0.8m) and betaine (2.5 mm), and in-
ducing bhp expression with IPTG (1 mm) for 18 h led to the
formation of high concentrations of Bhp as a soluble protein,
although cell growth was retarded by the presence of sorbi-
tol.[17]
Sequence comparison of Bhp
Comparison of the amino acid sequence derived from the nu-
cleic acid sequence of bhp with proteins found in data bases
had indicated that Bhp might be a halogenating enzyme of
the perhydrolase type, since Bhp has more than 40% identity
to BPO-A2 from Streptomyces aureofaciens, to CPO-T from
Streptomyces aureofaciens Tꢀ24 and to CPO-P from Pseudomo-
nas pyrrocinia.[5] These enzymes had originally been identified
as “haloperoxidases”, but were later found to belong to the a/
b-hydrolase family and their apparent halogenating activity to
be due to the formation of peracids that subsequently oxi-
dised halide ions in a nonenzymatic reaction to form hypoha-
lous acid as the halogenating agent.[5] It could be demonstrat-
ed that these perhydrolases were not involved in halogenation
reactions during halometabolite formation.[8]
Purification of Bhp and partial physical characterisation
Bhp was purified to homogeneity by immobilised nickel-che-
lating chromatography. The size of the enzyme, including the
his-tag epitope, was analysed by SDS-PAGE (35000 Da) and by
MALDI-TOF MS (34011Æ40 Da; Figure 1). These experimentally
Despite this high sequence identity between Bhp and perhy-
drolases, no halogenating activity was detected with Bhp upon
using monochlorodimedone and phenol red, which are com-
monly employed substrates for the detection of haloperoxi-
dase and perhydrolase activity.[9–10] This is in agreement with
the results of in vivo experiments by Puk et al.,[7] who showed
that the two chlorination reactions that occur during balhimy-
cin biosynthesis are catalysed by the flavin-dependent halo-
genase BhaA and not by Bhp. However, inactivation of Bhp
resulted in a loss of balhimycin production that could be
restored by the addition of chemically synthesised b-OH-Tyr.
These results of Puk et al.[7] showed that Bhp is involved in b-
OH-Tyr biosynthesis.
The very high sequence identities of Bhp to putative thioes-
terases such as ORF3 from Streptomyces sp (41% identity),
which is involved in streptothricin biosynthesis,[11] suggested
that Bhp might actually act as a thioesterase during the bio-
synthesis of the nonproteinogenic amino acid b-OH-Tyr and
catalyse the release of b-OH-Tyr from the peptidyl carrier pro-
tein BspD.[7] In this context, it is not surprising that the identity
to ORF18 found in the biosynthetic gene cluster of the struc-
turally related type-I glycopeptide antibiotic chloroeremomy-
cin, which also contains two b-OH-Tyr residues, is as high as
85%.[4] Hubbard and Walsh[12] had suggested that CepJ
Figure 1. SDS-PAGE and MALD MS of purified Bhp.
obtained masses match well with the calculated mass of
33993. The identity of the enzyme was verified by partial N-
terminal amino acid sequencing, which showed that the first
59 amino acids, including the 33 of the His-tag, were identical
to those derived from the gene. According to size-exclusion-
chromatography experiments, native Bhp exists as a homodi-
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267

K.-H. van Pꢀe et al.
mer. The isoelectric point of Bhp was determined to be 5.4–
5.6. Bhp is stable over a pH range of 6.0–8.5, and glycerol was
found to stabilise the enzyme. By using buffers containing
glycerol (10%), an approximately 1.7-fold higher concentration
of Bhp could be obtained than when using buffers without
glycerol.
Since Bhp did not show any halogenating activity with the
haloperoxidases/perhydrolase substrates monochlorodime-
done and phenol red; based on its high sequence identity
with esterases, substrates for nonspecific esterases and lacto-
nases such as p-nitrophenyl acetate, p-nitrophenyl phosphate,
S-thiophenyl acetate and dihydrocoumarin were tested as sub-
strates for Bhp. However, Bhp did not show any esterase or lac-
tonase activity with any of these compounds.
By using chemically synthesised b-OH-Tyr-SNAC as a sub-
strate mimic for peptidyl carrier protein-bound b-OH-Tyr, it
could be demonstrated that Bhp catalyses the hydrolysis of
the thioester bond. Thus it could be shown for the first time in
an in vitro experiment that a thioesterase is involved in balhi-
mycin biosynthesis. This fits nicely with the data obtained from
feeding and in vivo experiments, in which it could be shown
that a mutant lacking a functional Bhp gene only produced
balhimycin when chemically synthesised b-OH-Tyr was ad-
ded.[7] This strongly suggested that, in balhimycin biosynthesis,
peptidyl carrier protein-bound b-OH-Tyr cannot be transferred
to the peptide synthetase that attaches b-OH-Tyr to the grow-
ing peptide chain. Instead free b-OH-Tyr has to be activated by
adenylation and then be transferred to the peptide synthetase.
According to the sequence homology of Bhp with CepJ
(ORF18), it can be assumed that CepJ (ORF18) has the same
function in chloroeremomycin biosynthesis.
It could be shown that Bhp catalyses the hydrolysis of the
thioester bond of b-OH-Tyr-SNAC 15 times more efficiently
than that of Tyr-SNAC. This is a rather significant selectivity
when compared to the thioesterase NikP2 from nikkomycin
biosynthesis, for which only a modest (~twofold) selectivity for
b-OH-His over His was reported.[13] The synthesis of b-OH-Tyr
according to Bolhofer,[18] Bodanszky and Bodanszky[19] and its
subsequent conversion to the SNAC derivative[20] gave two dia-
stereomers (A and B) in a ratio of 1:0.16 that could be separat-
ed by analytical (Figure 2), but not by preparative HPLC. When
using a combination of the methods described by Guanti
et al.[21] and Weissman et al.[22] for the synthesis of b-OH-Tyr-
SNAC, the ratio of the diastereomers (A/B) was 1:2.39 (Fig-
ure 2B). On the basis of its larger coupling constant, form A
Figure 2. HPLC chromatograms of purified b-OH-Tyr-SNAC A) synthesised
according to Bolhofer,[18] Bodanszky and Bodanszky,[19] and Ehmann et al.[20]
and B) alternatively synthesized as described by Guanti et al.[21] and Weiss-
man et al.[22] The retention time of the anti-diastereomer of b-OH-Tyr-SNAC
(form A) is 10.17 min and that of the syn-diastereoisomer (form B) is
9.32 min. C) HPLC analysis of the hydrolysis of b-OH-Tyr-SNAC by Bhp. The
chromatogram of the reaction of purified Bhp with b-OH-Tyr-S-NAC is
shown as a full line and the control lacking purified Bhp is shown as a
dotted line. The elution of b-OH-Tyr was observed at 6.65 min.
was identified by 1H NMR as the anti-diastereomer (3J5H,6H
=
6.6 Hz compared to 3J5H,6H =4.4 Hz for the syn-diastereomer).
Interestingly, only the anti-diastereomer (form A) was accepted
as a substrate by Bhp (Figure 2C).
The Km values for the thioesterases involved in polyketide or
nonribosomal peptide biosynthesis are often difficult to deter-
mine, since the substrates cannot always be used in concentra-
tions above Km.[23–25] The rather high Km value is most likely due
to the fact that b-OH-Tyr-SNAC is not the natural substrate of
Bhp, but only a substrate mimic. Similarly high Km values have,
for example, been reported for thioesterases involved in eryth-
romycin (12.2 mm),[23] picromycin (7.3–15 mm),[24] tylosin (28.2–
37.9 mm)[25] and surfactin (5 mm) biosynthesis.[26] For the thio-
When the concentration of the syn-diastereomer (form B)
was increased in the assay mixture to 1:0.33 or 1:2.39, inhibi-
tion of Bhp was observed to a relative activity of 72% or 18%,
respectively, probably due to the inhibitory effect of the syn-
diastereomer. The kobs of Bhp with b-OH-Tyr-SNAC containing
14% of the syn-diastereomer (form B) was 1.7 minÀ1, and Km
was estimated to be 8.3 mm by nonlinear least-squares fit from
a substrate saturation curve (Figure 3).
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b-OH-Tyr-PCP Hydrolase
BglII, NdeI and EcoRI restriction sites were introduced by PCR with
the following primers: Hydsm+ containing a BglII restriction site
upstream of the start codon and a NdeI restriction site at the start
codon 5’-ATA TAG ATC TTC ATA TGC TGA TGA CGA CTG AGC ACG
GG-3’ and HydsmÀ containing an EcoRI restriction site at the stop
codon 5’-ATA TGA ATT CAC AGT TCT TCG GGG GTC C-3’ (restriction
sites are underlined and start and stop codons are marked with
bold letters). After extraction from agarose gels by the freeze–
squeeze method,[28] the PCR product was blunt-end-ligated into
the EcoRV site of pBluescript SKII+. The resulting plasmid, pSM-
HBm, was introduced into E. coli XL1 Blue by electroporation. pSM-
HBm was then purified by the E.Z.N.A. method (PEQLAB Biotechno-
logie GmbH) followed by sequencing with T7 and T3 primers. For
expression in E. coli, the clones containing the bhp gene were di-
gested with BglII/EcoRI, and the desired DNA fragment was ligated
into the BamHI/EcoRI sites of pRSET B to generate pSM-HR13.
Plasmid pSM-HR13 was introduced into E. coli BL21(DE3)pLysS by
using the calcium chloride method.[29]
Figure 3. Substrate saturation curve of the Bhp-catalysed hydrolysis of b-OH-
Tyr-SNAC.
esterase NikP2, which is involved in nikkomycin biosynthesis,
in vitro activity has also been shown. However, in this case, the
natural substrate, b-OH-His bound to the didomain NRPS
Purification of Bhp: HNB medium (20 mL) containing ampicillin
(110 mgmLÀ1) and chloramphenicol (40 mgmLÀ1) in a 50 mL Erlen-
meyer flask was inoculated with a glycerol stock of E. coli BL21-
(DE3)pLyS (5 mL) harbouring the plasmid pSM-HR13. After incuba-
tion for 7 (378C, 160 rpm) or 12 h (308C, 150 rpm) until an OD600 of
about 1.5 was reached, the cells were transferred to HNBBS
medium (1 L) containing the appropriate antibiotics in a 2 L Erlen-
meyer flask and incubated at 378C for about 3 h until the OD600 of
the culture reached 0.3–0.4. Cells were induced with IPTG (1 mm),
incubated for further 16 h (308C, 150 rpm) and subsequently har-
vested by centrifugation for 30 min (10808g, 48C). After being
washed with potassium phosphate buffer (0.1m, pH 7.0), the cells
(2 g) were resuspended in ice-cold potassium phosphate buffer
(0.1m, pH 7.0, 10 mL) and sonicated for 1 min (Branson digital soni-
fier 450, 65% amplitude, 5 s disruption, 10 s break, 28C). The
homogenate was centrifuged for 40 min (30600g, 48C), and the
crude extract was immediately dialysed against potassium phos-
phate buffer (0.1m, pH 7.0, 2 L) containing EDTA (1 mm) and glyc-
erol (10%) and stored at À208C in aliquots (2 mL). To purify the
enzyme, freshly prepared crude extract (3 mL) was dialysed over-
night against starting buffer [NaCl (0.15m), glycerol (10%) in potas-
sium phosphate buffer (10 mm, pH 7.0, 1.5 L)] and loaded onto a
nickel-chelating Sepharose fast-flow column (9 mL column
volume). The column was washed with 10 column volumes of
starting buffer, followed by a linear gradient of 20–30% elution
buffer [NaCl (0.15m), glycerol (10%), imidazole (0.325 m) in potassi-
um phosphate buffer (10 mm, pH 7.0)] for 7 column volumes.
Bound protein was eluted with 4 column volumes of 100% elution
buffer. The eluted protein was analysed by SDS-PAGE[30] by using
4% stacking and 10% resolving gels. Fractions containing the pro-
tein of interest (35 kDa) were collected and immediately dialysed
against storage buffer [NaCl (0.15m), glycerol (10%), EDTA (1 mm)
in potassium phosphate buffer (10 mm, pH 7.0)]. Aliquots of the
purified enzyme were stored at À208C for further analysis.
NikP1, was used. Due to the low activity, only
a kobs
(0.14 minÀ1) was reported, but no Km.[13] Bhp, like perhydrolases
and the thioesterases to which it shows a high sequence iden-
tity, contains a catalytic triad consisting of a serine, a histidine
and an aspartate residue; this suggests that Bhp could be a
serine hydrolase. This hypothesis is supported by the finding
that Bhp activity is inhibited by 28, 40 or 80% in the presence
of 1, 2 or 5 mm phenylmethylsulfonyl fluoride (PMSF), respec-
tively.
Conclusions
The identification of Bhp as a thioesterase catalysing the hy-
drolysis of the thioester bond of b-OH-Tyr-SNAC is the last
piece of evidence required to fully understand the formation
of b-OH-Tyr as a precursor in balhimycin and other b-OH-Tyr-
containing glycopeptide antibiotics. In balhimycin biosynthesis,
the gene products of the operon containing the three genes
bpsD, oxyD and bhp provide b-OH-Tyr in its free form, which is
then incorporated into the growing peptide chain as residues
2 and 6 of the heptapeptide.[27]
Experimental Section
Bacterial strains, vectors and culture conditions: pUC18-Pst7kb
harbouring a 7101 bp PstI fragment containing the hydrolase gene
bhp from the balhimycin biosynthetic gene cluster was used for
amplification of the bhp gene by PCR.[3] pSM-HBm, a pBluescript
SK II+ (Stratagene) derivative containing the bhp gene, was used
to sequence bhp, and pSM-HR13, a pRSET B (Invitrogen) derivative
containing the bhp gene, was employed in expression experi-
ments. E. coli XL1-Blue was grown in HNB medium [NaCl (5 gLÀ1),
peptone from meat (5 gLÀ1), yeast extract (5 gLÀ1) and meat ex-
tract (3 gLÀ1)]. HNBBS (HNB-medium containing 0.8m d-sorbitol
and 2.5 mm betaine-HCl) was used for the expression of bhp from
the plasmid pSM-HR13 in E. coli BL21(DE3)PlysS.
Characterisation of physical properties of Bhp: Protein concen-
trations were determined according to Whitaker and Granum.[31]
The identity of the enzyme was verified by partial N-terminal
amino acid sequencing by automated Edman degradation. The
molecular weight of the Bhp subunits was determined by SDS-
PAGE, as described above, and by MALDI-TOF MS analysis. To de-
termine the native molecular weight of Bhp, a HiLoad 16/60 Super-
dex 200 column (Pharmacia) equilibrated with NaCl (0.15m), glycer-
ol (10%) and potassium phosphate buffer (10 mm, pH 7.0), was
DNA manipulation, construction of plasmids and expression of
bhp: bhp was amplified from the plasmid pUC18-Pst7kb by PCR.
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K.-H. van Pꢀe et al.
used with the Bio-Rad gel filtration standard. Eluted fractions were
analysed by SDS-PAGE to determine the elution time of Bhp.
supernatants were analysed by HPLC, as described above. All reac-
tions were performed in duplicate, and the background activity
due to chemical hydrolysis was determined by using dialysis buffer
instead of enzyme.
The isoelectric point (pI) of Bhp was determined by isoelectric fo-
cussing under denaturing conditions according to the 2D Electro-
phoresis Handbook from Amersham Biosciences using the Ettan
IPGphor IEF system and immobilised pH gradient dry strip gels
from Amersham Biosciences with a length of 18 cm at a pH range
between pH 3–10. The pI of Bhp was determined by comparison
with IEF marker from Amersham Bioscience.
Synthesis of l-Tyr-SNAC: l-Tyr-SNAC was synthesised according to
the general procedure for synthesis of aminoacyl-SNAC derivatives
described by Ehmann et al.[20] starting from N-tert-butyloxycarbonyl
l-tyrosine (Boc-l-Tyr). The Boc-protecting group was removed by
treatment with TFA (50%) in dichloromethane. The identity of the
product was confirmed by HPLC, HPLC-MS and NMR analysis.
Enzyme assays: Halogenating activity was analysed according to
the monochlorodimedone assay, as described by Hewson and
Hager[9] and with phenol red as the substrate for activity staining
after native PAGE.[10]
Synthesis of b-OH-Tyr-SNAC: b-OH-Tyr-SNAC was synthesised
from N-tert-butyloxycarbonyl-b-hydroxytyrosine (Boc-(b-OH-Tyr))
and NAC in analogy to the preparation of l-Tyr-SNAC. Boc-(b-OH-
Tyr) was synthesised from p-benzyloxybenzaldehyde and glycine
by using a combination of the methods described by Bolhofer[19]
and Bodanszky and Bodanszky.[19] Boc-(b-OH-Tyr) was then treated
with NAC using the coupling reagents DCC/HOBt to produce Boc-
(b-OH-Tyr)-SNAC, as described above for Boc-Tyr-SNAC. Purification
of Boc-(b-OH-Tyr)-SNAC was carried out by preparative HPLC on a
glass column containing silica gel and chloroform/methanol (93:7)
as the mobile phase. Fractions containing Boc-(b-OH-Tyr)-SNAC,
which were detected by TLC, were collected, and the solvent was
removed by rotary evaporation. The residue was then analysed by
HPLC, HPLC-MS and NMR. b-OH-Tyr-SNAC, which was obtained
after cleavage of the protecting group as described above for l-
Tyr-SNAC, was purified by preparative HPLC with a Eurospher-100
C18 column (5 mm, 20ꢄ250 mm). Separation was performed by
isocratic elution with a flow rate of 7.5 mLminÀ1 at room tempera-
ture with acetonitrile/water/acetic acid (16:84:0.5) as the solvent.
The two diastereoisomers of b-OH-Tyr-SNAC were not resolved
well enough for preparative separation. Fractions containing b-OH-
Tyr-SNAC were identified by analytical HPLC and pooled, and the
solvent was removed by freeze drying. The white residue of b-OH-
Tyr-SNAC was analysed by HPLC and HPLC-MS, and stored immedi-
ately at À208C. The identity of the product was established by
HPLC-MS and NMR analysis. Alternatively, b-OH-Tyr was synthesised
according to Guanti et al.[21] and converted to the SNAC derivative
as described by Weissman et al.[22]
Hydrolase-activity assays were performed spectrophotometrically
by using dihydrocoumarin, p-nitrophenyl acetate, p-nitrophenyl
phosphate and S-thiophenyl acetate as the substrates according to
Casellas et al.,[32] Rowe and Howard,[33] Zhuo et al.[34] and Bergmey-
er,[35] respectively.
Thioesterase activity assay with N-acetyl cysteamine derivatives:
The activity assays of Bhp with S-b-hydroxytyrosyl-N-acetyl cystea-
mine thioester (b-OH-Tyr-SNAC) and S-l-tyrosyl-N-acetyl cysteamine
thioester (l-Tyr-SNAC) as substrates were carried out in a combina-
tion of the assays described by Trauger et al.[36] and Boddy et al.[37]
with some modifications. The reaction mixtures (75 mL) containing
purified Bhp (10 mL, 5.3 mg) and substrate (2 mm) in potassium
phosphate buffer (50 mm, pH 7.0) were incubated at 308C for
45 min. The reactions were stopped by adding trifluoroacetic acid
(TFA; 2.5 mL, 20%, v/v) in H2O, the mixture was centrifuged, and
the supernatants were stored at À208C. For HPLC analysis, samples
were thawed, and methanol (19 mL) was added. After mixing, the
samples were centrifuged for 1 min (16100g, RT), and the superna-
tant (20 mL) was injected. HPLC analysis was carried out on a Euro-
spher-100 C18 (5 mm, 4ꢄ250 mm) column with methanol/water/
TFA (20:80:0.1) as the mobile phase. Elution was performed at a
flow rate of 0.4 mLminÀ1 with isocratic elution and monitored at
280 nm for 40 min.
To determine the kinetic parameters, the assay mixtures (305 mL)
contained purified Bhp (45 mL, 23.8 mg) and substrates at concen-
trations of 0.65, 1.29, 1.94, 2.58, 3.23, 4.52, 5.8 and 6.45 mm in po-
tassium phosphate buffer (50 mm, pH 7.0). Higher substrate con-
centrations could not be used due to solubility restrictions of the
substrate in water. The reactions were initiated by the addition of
enzyme and incubated at 308C. At intervals of 0, 3, 7, 15, 30 and
45 min, samples (75 mL) were taken, and the reaction was stopped
by adding TFA in H2O (20%, v/v, 2.5 mL), the mixture was centri-
fuged, and the supernatants were analysed by HPLC as described
above. 1 unit of thioesterase activity was defined as the formation
of 1 mmol b-OH-Tyr per min. All reactions were performed in dupli-
cate, and the background reaction due to chemical hydrolysis was
determined by using dialysis buffer instead of enzyme and sub-
tracted from the activity obtained in the assay of the enzymatic
reaction. Determination of the kinetic data for Tyr-SNAC was not
possible due to the very low activity of Bhp with this substrate.
Acknowledgements
We wish to thank BASF AG for N-terminal amino acid sequencing
and MALDI-TOF MS analysis of Bhp. We gratefully acknowledge a
grant to S.M. within the frame of the Development of Undergrad-
uate Education (DEU) Project of the LPIU Universitas Sebelas
Maret (Indonesia) from The World Bank in cooperation with the
Deutsche Akademische Austauschdienst (DAAD). This work was
also supported by the Deutsche Forschungsgemeinschaft (DFG Pe
438/16–1 and 16–2, DFG Su 239/3–3) and the EU (COMBIG-TOP–
LSHB-CT-2003-503491) to W.W. and R.D.S.
Keywords: antibiotics · balhimycin · beta-hydroxytyrosine ·
glycopeptides · thioesterases · vancomycin
The effect of PMSF on the Bhp-catalysed reaction was determined
under the following conditions: purified Bhp (22 mL, 20 mg) and
PMSF at concentrations of 0, 1, 2 and 5 mm were incubated for
15 min, and then potassium phosphate buffer (50 mm, pH 7.0,
58 mL) containing b-OH-Tyr-SNAC (2.6 mm) was added. The assays
were incubated at 308C for 30 min and then stopped by adding
TFA in H2O (20%, v/v, 2.5 mL), the mixture was centrifuged, and the
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Published online on December 8, 2009
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