Báo cáo khoa học: Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis pdf

Mutational analysis of a type II thioesterase associated
with nonribosomal peptide synthesis
Uwe Linne, Dirk Schwarzer, Gunnar N. Schroeder* and Mohamed A. Marahiel
Philipps Universita
¨
t Marburg, Fachbereich Chemie/Biochemie, Germany
Recent studies on type II thioesterases (TEIIs) involved in
microbial secondary metabolism described a role for these
enzymes in the removal of short acyl-S- phosphopantetheine
intermediates from misprimed holo-(acyl carrier proteins)
and holo-(peptidyl carrier proteins) of polyketide synthases
and nonribosomal peptide synthetases. Because of the
absence of structural information on this class of enzymes,
we performed a mutational analysis on a prototype TEII
essential for efficient production of the lipopeptide antibiotic
surfactin (TEII
srf
), which led to identification of catalytic and
structural residues. On the basis of sequence alignment of 16
TEIIs, 10 single and one double mutant of highly conserved
residues of TEII
srf
were constructed and biochemically
investigated. We clearly identified a catalytic triad consisting
of Ser86, Asp190 and His216, suggesting that TEII
srf
belongs
to the a/b-hydrolase superfamily. Exchange of these residues
with residues with aliphatic side chains abolished enzyme
activity, whereas replacement of the active-site Ser86 with
cysteine produced an enzyme with marginally reduced

activity. In contrast, exchange of the second strictly con-
served asparagine (Asp163) with Ala resulted in an active but
unstable enzyme, excluding a role for this residue in catalysis
and suggesting a structural function. The results define three
catalytic and at least one structural residue in a nonribo-
somal peptide synthetase TEII.
Keywords: catalytic triad; fatty acid synthases; nonribosomal
peptide synthesis; peptide synthetases; type II thioesterase
polyketide synthases.
Enzymes that cleave thioesters are ubiquitous in prokary-
otes and eukaryotes, as thioesters appear in many different
metabolic processes. For example, thioesterases have been
reported to cleave formate from formylated glutathione [1],
which is an intermediate in formaldehyde detoxification
in plants, and fatty acids from cysteines of lipidated
proteins [2].
Most common thioesterases are involved in 4¢-phospho-
pantetheine (4¢-Ppant) metabolic processes, such as the
synthesis of fatty acids, polyketides, or nonribosomal
peptides. Many polyketides and nonribosomal polypeptides
produced by bacteria and filamentous fungi are of great
pharmacological interest. Among these are molecules that
exhibit antibiotic (penicillin, cephalosporin, erythromycin
and vancomycin), immunosuppressive (cyclosporin) and
cytostatic (bleomycin and epothilone) activities. A common
feature is that they are biosynthesized by large modular
enzymes, the so called nonribosomal peptide synthetases
(NRPSs) and the polyketide synthases (PKSs) [3,4]. During
synthesis, all substrates and intermediates are covalently
tethered to the enzymatic templates through a thioester

linkage [5]. The thiol group of this thioester belongs to
4¢-Ppant, the prosthetic group of the peptidyl carrier
proteins (PCPs) and acyl carrier proteins. The post-trans-
lational modification (priming) of the carrier proteins is
carried out by dedicated 4¢-phosphopantetheine transferases
such as Sfp [6–8].
Two types of thioesterase are associated with NRPSs
and PKSs: the well-studied integrated type I thioesterase
domains (TE domains), which are responsible for the release
of the synthesized products from the enzymatic templates
[9–11], and the external stand-alone type II thioesterases
(TEIIs). Disruption of the corresponding TEII genes in the
producer strains inhibited product formation by 80–90%
[12–14]. Recently, biochemical studies on TEIIs in polyke-
tide synthesis suggested a role in the removal of short acyl
chains originating from aberrant decarboxylation of chain
extender units from the thiol moiety of the 4¢-Ppant
cofactors of acyl carrier proteins [15,16]. In NRPSs there
is no such decarboxylation process during product synthe-
sis. However, we recently demonstrated that TEIIs associ-
ated with NRPSs are also involved in the regeneration of
misprimed PCPs by removing short acyl chains from the
4¢-Ppant cofactors [17]. These acyl chains are thought to be
transferred to NRPSs during the priming process, because
acyl-CoAs, which are present in significant concentration in
Correspondence to M. A. Marahiel, Philipps Universita
¨
t Marburg,
Fachbereich Chemie/Biochemie, Hans-Meerwein-Strasse,
35032 Marburg, Germany.

Fax: + 49 6421 2822191, Tel.: + 49 6421 2825722,
E-mail:
Abbreviations: DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); NRPS,
nonribosomal peptide synthetase; PCP, peptidyl carrier protein;
Ppant, phosphopantetheine; PKS, polyketide synthase; T, thiolation
domain, referring to the same thing as PCP, but used for the des-
cription of proteins (Ôone letter–one domainÕ nomenclature of NRPSs);
TEII, type II thioesterase; TNB, 5-thio-2-nitrobenzoic acid;
ESI, electrospray ion.
*Present address: Institute of Microbiology, ETH-Zu
¨
rich,
Schmelzbergstr.7, Zu
¨
rich, Switzerland.
(Received 5 January 2004, revised 16 February 2004,
accepted 2 March 2004)
Eur. J. Biochem. 271, 1536–1545 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04063.x
the cells under most growth conditions [18], can also be
utilized as substrates by 4¢-phosphopantetheinyl trans-
ferases [6].
Interestingly, TEIIs associated with microbial secondary
metabolism also show remarkably high sequence similarities
(> 20%) and length ( 240–260 residues) to a specialized
mammalian (rat) TEII (TEII
rat
), which is involved in fatty
acid biosynthesis in specialized tissues such as the mammary
gland [12,19]. There, it catalyzes the release of short-chain
fatty acids which are ingredients of milk [20]. In the case of

TEII
rat
, two residues (Ser101, His237) have been suggested to
be part of a catalytic triad [21–23]. Asp236 has been reported
to enhance the catalytic activity of the enzyme, although its
role in catalysis remains unclear as the catalytic efficiency of
the Ala mutant was only marginally reduced (by  40%).
Therefore, it is not clear if a catalytic triad or a catalytic diad
consisting of only Ser and His is required for catalysis.
In this study, we describe a mutational analysis and
mechanistic investigations of the microbial TEII
Srf
(242
amino acids,  28 kDa), which is associated with the
production of the secondary metabolite surfactin [12,24].
A total of 11 TEII
Srf
mutants, including mutations of all
strictly conserved residues with functionalized side chains
among 16 TEIIs of diverse origin, were generated by site-
directed mutagenesis and biochemically characterized to
define important catalytic and structural residues and
to determine if these enzymes are mechanistically related
to TEII
rat
of primary metabolism.
Experimental procedures
Sequence alignment to identify highly conserved
residues
The sequences of 16 TEIIs were retrieved from the pub-

licly accessible NCBI database ( />Sequences used were derived from NRPS and PKS
biosynthetic clusters, in addition to that of the mammalian
TEII
rat
. The sequences ( 250 amino-acid stretches) were
aligned using the program
MEGALIGN
from the DNA Star
package, applying the method of Jotun Hein with default
parameters.
Construction of TEII
srf
and TEII
srf
mutant expression
plasmids and protein purification
Cloning and overproduction of wild-type TEII
srf
has been
described previously [17]. All mutants generated in this
study were introduced into plasmid pTEII
srf
[17]. Mutants
were obtained using the Quick Change Site Directed
Mutagenesis Kit
TM
(Stratagene, Heidelberg, Germany) as
described by manufacturer. The mutants constructed and
the primers used with plasmid pTEII
srf

as template are
summarized in Table 1. All mutant plasmids generated were
confirmed by DNA sequencing using the ABI Prism
dRhodamine Terminator Cycle Sequencing Ready Reac-
tion Kit (v3.0) and an ABI 310 DNA sequencer (both
Applied Biosystems, Darmstadt, Germany) according to
the manufacturer’s protocols.
Escherichia coli M15/pREP4 was transformed separately
with all mutant plasmids constructed. Expression and
purification of the His
6
-tagged enzymes (except D163A)
were performed as previously described for wild-type TEII
srf
[17]. In the case of D163A, an overnight culture of the
expression strain was grown at 22 °C and induced with iso-
propyl thio-b-
D
-galactoside (0.2 m
M
). After an additional
3 h incubation at 22 °C, the cells were centrifuged at 4 °C,
and D163A was then purified, dialyzed against assay buffer
using HiTrap
TM
Desalting columns (Amersham Biosciences,
Freiburg, Germany) according to the manufacturer’s pro-
tocols, and assayed for activity.
Overproduction and purification of the enzymes after
single-step Ni

2+
-affinity chromatography were confirmed
by SDS/PAGE [25]. Protein concentrations were assigned
Table 1. TEII
srf
mutants constructed and primers used (mutations introduced are underlined).
Mutation Primer Primer sequences (5¢ and 3¢)
TEII
srf
C18A 5¢ CA CAG CTC ATC GCT TTT CCG TTT GCC GGC GGC
3¢ GCC GCC GGC AAA CGG AAA AGC GAT GAG CTG TG
TEII
srf
H85A 5¢ GTG CTG TTC GGA GCC AGT ATG GGC GGA ATG ATC AC
3¢ GT GAT CAT TCC GCC CAT ACT GGC TCC GAA CAG CAC
TEII
srf
S86A 5¢ G CTG TTC GGA CAC GCT ATG GGC GGA ATG ATC ACC
3¢ GGT GAT CAT TCC GCC CAT AGC GTG TCC GAA CAG CAC
TEII
srf
S86C 5¢ GTG CTG TTC GGA CAC TGT ATG GGC GGA ATG ATC AC
3¢ GT GAT CAT TCC GCC CAT ACA GTG TCC GAA CAG CAC
TEII
srf
S112A 5¢ G GCG GTT ATC ATT GCT GCA ATC CAG CCG CC
3¢ GG CGG CTG GAT TGC AGC AAT GAT AAC CGC C
TEII
srf
D163A 5¢ G CCT TCT TTC CGA TCA GCT TAC CGG GCT CTT G

3¢ C AAG AGC CCG GTA AGC TGA TCG GAA AGA AGG C
TEII
srf
D189A 5¢ C TTT AAC GGG CTT GCT GAT AAA AAA TGC ATA CGA GAT GCG G
3¢ C CGC ATC TCG TAT GCA TTT TTT ATC AGC AAG CCC GTT AAA G
TEII
srf
D190A 5¢ C TTT AAC GGG CTT GAT GCT AAA AAA TGC ATA CGA GAT GCG G
3¢ C CGC ATC TCG TAT GCA TTT TTT AGC ATC AAG CCC GTT AAA G
TEII
srf
5¢ C TTT AAC GGG CTT GCT GCT AAA AAA TGC ATA CGA GAT GCG G
DD189/190AA 3¢ C CGC ATC TCG TAT GCA TTT TTT AGC AGC AAG CCC GTT AAA G
TEII
srf
H216L 5¢ CAA TTT GAC GGC GGG CTC ATG TTC CTG CTG TC
3¢ GA CAG CAG GAA CAT GAG CCC GCC GTC AAA TTG
Ó FEBS 2004 Mechanistic studies on TEII
srf
(Eur. J. Biochem. 271) 1537
using UV spectroscopy (A
280
). The calculated absorption
coefficients of wild-type TEII
srf
and TEII
srf
mutants are very
similar. The value of the wild-type TEII
srf

(16 860
M
)1
Æcm
)1
)
was used for calculation of all enzyme concentrations. After
addition of 10% (v/v) glycerol, enzymes were shock-frozen
in liquid nitrogen and could be stored at )80 °C over several
weeks without significant loss of activity, except TEII
srf
-
D163A, which seems to be unstable and was directly assayed
after expression and purification.
Enzymatic preparation of acetyl-
S
-4¢-Ppant-PCP
The preparation of acetyl-S-Ppant-PCP, which was used as
substrate for TEII
srf
and TEII
srf
mutants, was carried out
according to previously developed protocols [17].
5,5¢-Dithiobis(2-nitrobenzoic acid) (DTNB)-HPLC assay
TEII
srf
or TEII
srf
mutants at a concentration of 25 n

M
were
incubated at 37 °C with various concentrations of acetyl-
S-Ppant-PCP (2–35 l
M
inthecaseofactivemutants;initial
concentration for activity test of all mutants was 25 l
M
)in
the presence of DTNB (8 lLofa10m
M
stock solution) in
assay buffer (50 m
M
Hepes, 100 m
M
NaCl, 1 m
M
EDTA,
10 m
M
MgCl
2
,pH7.0)inatotalvolumeof400lL.
At defined time points, 50 lL samples were collected, and
the reaction was subsequently stopped by the addition of
100 lL methanol/trifluoroacetate (500 : 1, v/v). The ratio of
acetyl-S-Ppant-PCP to 5-thio-2-nitrobenzoic acid (TNB)-
S-Ppant-PCP was analyzed by the HPLC method described
previously [17].

Assay of [1-
14
C]acetyl-
S
-Ppant-enzyme hydrolysis
Apo-PCP [17] was incubated at a concentration of 1 m
M
in a total volume of 1.3 mL with 20 l
M
[1-
14
C]acetyl-CoA
(50 mCiÆmmol
)1
;ICN,Eschwege,Germany),50n
M
Sfp,
and 10 m
M
MgCl
2
in assay buffer (50 m
M
Hepes, 100 m
M
NaCl, 1 m
M
EDTA, pH 7.3) at 37 °C. After complete
modification, the reaction mixture was split and TEII
srf

or TEII
srf
mutants were added to one aliquot to a final
concentration of 500 n
M
inthecaseofPCP.Samples
(100 lL) were taken at defined time points and mixed with
800 lL trichloroacetic acid (10%, w/v) and 15 lLBSA
solution (25 mgÆmL
)1
). Denatured proteins were collected
by centrifugation, washed with 800 lL 10% (v/v) trichloro-
acetic acid, and dissolved in 400 lL formic acid. Enzyme-
bound radioactivity was analyzed by liquid scintillation
counting (Tri-Carb 2100 TR, Packard, Germany).
Proline quench assay
Holo-ProCAT [17] was incubated at a concentration of
1 l
M
in a total volume of 1.5 mL with 10 m
M
MgCl
2
,5 m
M
ATP and 4.1 l
M
[
14
C]Pro (246 mCiÆmmol

)1
; Hartmann
Analytics, Braunschweig, Germany) in assay buffer at
37 °C. After complete aminoacylation, 15 lL of a 100 m
M
unlabeled proline solution was added, and the reaction
mixture was split. TEII
srf
or TEII
srf
mutants were added to
one aliquot to a final concentration of 500 n
M
. At defined
time points, 100 lL samples were removed and prepared
and analyzed as described above.
TNB modification of TEII
srf
and TEII
srf
mutants
DTNB (4 lLofa10m
M
solution in dimethyl sulfoxide)
was added to 46 lLofa25l
M
solution of TEII
srf
,
TEII

srf
C18A, TEII
srf
S86C or TEII
srf
S86A and incubated
for 30 min at room temperature. Subsequently, the yellow
reaction mixture was applied to MicroSpin
TM
G-50 col-
umns (Amersham Biosciences), which were pre-equilibrated
with assay buffer (2 · resuspension in 400 lL assay buffer
followed by centrifugation at 735 g). The colorless enzyme
solutions were collected in fresh tubes by centrifugation of
the columns at 735 g. Samples were desalted by application
to a 30/2 mm Nucleosil C8 column (Macherey-Nagel,
Du
¨
ren, Germany) using an Agilent 1100 Series HPLC
system (Agilent Technologies, Waldbronn, Germany).
The following gradient was applied at a flow rate of
0.1 mLÆmin
)1
[buffer A: 0.1% (v/v) trifluoroacetate in
water; buffer B: 0.1% (v/v) trifluoroacetate in acetonitrile]:
holding buffer B constant (10%) for 4 min, followed by a
linear gradient to 95% buffer B in 1 min and elution of the
enzymes at 95% buffer B for 10 min. Samples were directly
transferred to an electrospray ion (ESI) source connected to
a Qstar Pulsar i mass spectrometer (Applied Biosystems,

Darmstadt, Germany). ESI-TOF spectra (300–3000 amu)
were recorded with the following parameters: curtain gas 25;
nebulizer gas 35; DP1 90 V; DP2 15 V; FP 220 V; ion spray
voltage 5500 V. Deconvolution was performed using the
supplied
ANALYST
TM
software.
CD spectroscopy of TEII
srf
mutants
For CD spectroscopy, TEII
srf
and TEII
srf
mutants were
dialyzed against 50 m
M
sodium phosphate buffer (pH 7;
19.5 m
M
NaH
2
PO
4
and 30.5 m
M
Na
2
HPO

4
) and diluted
in the same buffer to a final concentration of 10 l
M
.
Spectra were recorded on a J-810 spectrapolarimeter
(Jasco, Grob-Umstadt, Germany). For this, 300 lL
enzyme solution was pipetted into a cuvette of 1 mm
layer thickness. The ellipticity was then measured at a
constant temperature of 25 °C in the wavelength range
260–190 nm and a scan speed of 50 nmÆmin
)1
(one data
point per nm). The bandwidth was set to 4 nm and the
response time was 4 s. Each sample was measured 10
times, and a final spectrum was calculated by the supplied
Jasco software. The data were evaluated by the method of
Yang [26].
Results
Homology searches to identify the target residues
for site-directed mutagenesis of TEII
srf
To identify strictly conserved residues among TEIIs
involved in microbial secondary metabolism, 15 amino-acid
sequences of NRPS and PKS TEIIs as well as the
mammalian TEII
rat
were aligned. A catalytic role for
Ser101, Asp236 and His237 in TEII
rat

has been reported
[21]. The relative identity scores between TEII
srf
and the
other TEIIs range from 20.3% (MegH [27] and TEII
rat
[19])
to 28.9% (TycF [28]) (Table 2). Two exceptions are
LchA-TE (59.1%) [29] and LicTE (58.3%) [30], which are
closely related to TEII
srf
. Interestingly, the overall identity
1538 U. Linne et al.(Eur. J. Biochem. 271) Ó FEBS 2004
compared with type I thioesterases (TE domains) [31] of
NRPSs is only about 10% (data not shown).
From the TEIIs investigated, we identified 18 absolutely
invariant residues, of which six (His85, Ser86, Ser112,
Asp163, Asp190 and His216) carry functionalized side
chains (carboxyl, amino, amine, guanidino, thiol or hydroxy
groups). Interestingly, Asp236 of TEII
rat
,whichwas
previously thought to be involved in catalysis in the case
of the mammalian TEII associated with primary metabo-
lism, is not conserved among the microbial TEIIs of
secondary metabolism (Fig. 1). Mutants of these six invari-
ant residues were generated, in which the functionalized
residues were exchanged with nonfunctionalized residues
(H85A, S86A, S112A, D163A, D190A and H216L). In
addition, Ser86, thought to be the active site Ser because it is

embedded in a typical core sequence found for many
hydrolases (GxSxG) [32] and because of comparison with
TEII
rat
, was replaced with Cys (S86C). In the case of active-
site serines of catalytic triads, it is known that such a
substitution slows down reactions, possibly allowing detec-
tion of covalent reaction intermediates [22]. His216, which is
the corresponding residue in TEII
srf
to the active-site His237
of TEII
rat
[23] and is located in a thioesterase core motif
(GxxHxF), was also replaced by Arg (H216R). Directly
adjacent to the invariant Asp190, a second Asp residue was
identified (Asp189). Therefore, a single (D189A) and double
(D189A/D190A) mutant were generated, ensuring that
these two residues cannot replace each other. Finally Cys18,
which is conserved in 14 out of the 16 TEIIs [in the other
two cases (NrpT and YbtT), a Cys is found in the close
neighborhood of this position], was changed to Ala (C18A),
because Witkowski & Smith [33] showed inhibition of
TEII
rat
with DTNB. DTNB is a reagent that modifies free
thiol groups [34]. They postulated that this modified Cys
residue is probably involved in substrate binding. Three Cys
residues are found in TEII
srf

and four in TEII
rat
, whereby
only the one mutated (Cys18 in TEII
srf
) is conserved.
Furthermore, the assay used for the kinetic characterization
of TEII
srf
depends on DTNB [17].
The most important parts of the alignment showing all
the conserved regions in which mutations were introduced
are illustrated in Fig. 1.
Generation, expression and purification of the TEII
srf
mutants
We constructed a set of 10 single TEII
srf
mutants (C18A,
H85A, S86A, S86C, S112A, D163A, D189A, D190A,
H216L and H216R; Fig. 1) and one double mutant
(DD189/190AA) by site-directed mutagenesis. Mutations
other than to alanine or leucine were designed to show
residual activity for similar functional groups (S86C and
H216R). In the case of a catalytic triad (Asp-His-Ser)
similar functionalized groups were expected to exhibit
residual activity, whereas nonfunctionalized groups would
have none. The integrity of all the mutants was confirmed
by DNA sequencing, and all were individually expressed as
C-terminal His

6
tag fusions in the heterologous host E. coli.
Table 2. Similarities (%) between the 16 aligned TEIIs. Associated biosynthesis operons: TEII
srf
, surfactinA [24]; BacT, bacitracin [39]; Ery ORF5,
erythromycin [40]; GrsT, gramicidin S [41]; LchA-TE, lichenysin D [29]; LicTE, lichenysin D [42]; MegH, megalomicin [27]; NrpT [43], NysE,
nystatin [44]; PchC, pyochelin [45]; PikAV, pikromycin [46]; PimI, piramicin [47]; RifR, rifamycin [48]; TycF, tyrocidin [28]; YbtT, yersinabactin
[49]; TEII
rat
, fatty acid synthase [19].
Part 1 TEII
srf
BacT Ery ORF5 GrsT LchA-TE LicTE MegH
TEII
srf
26.5 23.6 24.0 59.1 58.3 22.3
BacT 23.1 31.6 25.2 24.4 23.5
Ery ORF5 31.6 26.7 25.9 77.7
GrsT 20.8 20.7 31.6
LchA-TE 97.3 25.5
LicTE 24.7
MegH
Part 2 NrpT NysE PchC PikAV PimI RifR TycF YbtT TeII
rat
TEII
srf
23.1 24.8 26.9 24.0 28.5 25.6 28.9 27.7 22.3
BacT 23.9 25.2 27.8 29.5 27.8 31.2 34.2 23.1 27.8
Ery ORF5 21.9 38.9 39.7 36.8 41.3 43.7 25.4 29.1 24.7
GrsT 22.7 32.7 27.1 31.6 31.4 32.4 29.9 25.8 27.0

LchA-TE 21.6 26.3 28.3 27.5 28.2 26.7 28.3 23.5 21.6
LicTE 21.4 23.9 27.5 24.5 26.3 25.7 26.6 23.7 21.0
MegH 22.7 36.8 38.1 38.5 36.4 41.3 26.6 27.1 24.3
NrpT 21.9 25.5 26.5 23.5 24.1 24.6 38.9 25.3
NysE 39.4 43.8 69.3 50.6 28.7 25.9 23.1
PchC 37.1 37.8 41.8 27.9 29.5 25.5
PikAV 48.6 42.9 29.9 32.6 22.4
PimI 49.8 29.9 27.8 25.9
RifR 29.1 27.0 26.3
TycF 24.6 24.2
YbtT 22.1
TeII
rat
Ó FEBS 2004 Mechanistic studies on TEII
srf
(Eur. J. Biochem. 271) 1539
Expression and purification was carried out as described
previously for wild-type TEII
srf
[17]. Because D163A was
unstable when expressed at 30 °C under standard condi-
tions and precipitated when dialyzed against 50 m
M
sodium
phosphate buffer (the others did not; see the paragraph
about CD spectroscopy of the TEII
srf
mutants), the
expression was performed at a lower temperature (22 °C).
As judged by SDS/PAGE, all His

6
-tagged recombinant
proteins were purified to near-homogeneity by single-step
Ni
2+
-affinity chromatography (data not shown).
Determination of the catalytic activities of the mutants
by the DTNB-HPLC assay
For the initial activity test, the previously developed
DTNB-HPLC assay was used [17]. In this assay, acetyl-
S-Ppant-PCP was used as a model substrate for wild-type
TEII
srf
, which is hydrolyzed very efficiently by the enzyme
(K
m
¼ 0.9 ± 0.4 l
M
; k
cat
¼ 95 ± 5 min
)1
[17]). The
products formed are HS-Ppant-PCP (holo-PCP) and
acetic acid. The products were analysed by an HPLC
method that requires modification of the free thiol of the
HS-holo-PCP with DTNB, resulting in TNB-holo-PCP.
Therefore, DTNB had to be added to the reaction
mixture. TNB-holo-PCP, acetyl-holo-PCP and apo-PCP
can be separated by an optimized HPLC method [17]. For

the initial activity screen, the PCP substrate was used at
a concentration of 25 l
M
, which is significantly higher
than the K
m
of TEII
srf
(0.9 ± 0.4 l
M
[17]). Mutants
C18A, H85A, S112A and D189A were still hydrolytically
active, whereas S86A, D190A, DD189/190AA, H216L
and H216R showed no hydrolytic activity in the 30 min
reaction time. The S86C mutant was inactive in the
DTNB-HPLC assay also, as shown in Fig. 2A. Further
biochemical characterization of this mutant is described
below. In initial assays directly after purification, mutant
D163A seemed to be active. However, the results could
not be reproduced with the same enzyme preparation
stored for a few days at )80 °C. Furthermore, as
mentioned above, it precipitated during dialysis against
sodium phosphate buffer. Therefore, the enzyme was
expressed at a lower temperature (22 °C), purified
(standard procedure), and dialyzed against assay buffer
directly after harvesting of the cells, avoiding the freezing
step. The enzyme was then subjected to the DTNB-HPLC
assay without storage for longer than 1 h on ice after the
purification procedure was finished. The D163A mutant
was then hydrolytically active towards its cognate sub-

strate acetyl-S-Ppant-PCP. Interestingly, after 5–10 min of
incubation at 37 °C, no further hydrolysis of the remain-
ing substrate was observed, indicating clear instability of
this enzyme under the assay conditions (data not shown).
However, the same enzyme preparation was still active
after storage of the stock solution for 24 h on ice.
To determine the effect of the mutations on enzyme
activity, a kinetic characterization of the active mutants
was performed according to Michaelis–Menten. The kin-
etic parameters obtained are summarized in Table 3 and
represent the results of at least three independent meas-
urements. As mentioned above, mutant S86C was not
suitable for the DTNB-HPLC assay, and D163A was
unstable during the assay. Therefore no kinetic parameters
could be determined for these mutants. The K
m
values
obtained for C18A (1.7 ± 0.4 l
M
), S112A (0.2 ± 0.7 l
M
)
and D189A (0.6 ± 1.2 l
M
) were in the same range as the
wild-type K
m
(0.9 ± 0.4 l
M
[17]). The same was true for

k
cat
(C18A 99 ± 4 min
)1
, S112A 98 ± 10 min
)1
, D189A
154 ± 18 min
)1
, and wild-type 95 ± 5 min
)1
) and there-
fore for k
cat
/K
m
(C18A 0.97 · 10
6
M
)1
Æs
)1
, S112A
8.2 · 10
6
M
)1
Æs
)1
, D189A 4 · 10

6
M
)1
Æs
)1
, and wild-type
1.75 · 10
6
M
)1
Æs
)1
). Obviously, the single mutations intro-
duced in these strictly conserved positions (C18A, S112A
Fig. 1. Alignment of 16 TEIIs. TEII
srf
was aligned together with 14 other TEIIs of microbial secondary metabolism. The mammalian TEII
rat
was
also added to the alignment. All the highly conserved regions are shown as well as the residues where mutations were introduced (marked by an
arrow).
1540 U. Linne et al.(Eur. J. Biochem. 271) Ó FEBS 2004
and D189A) had only a minor effect on the catalytic
efficiency of the enzyme. The K
m
of mutant H85A
(0.5 ± 0.03 l
M
) was also very close to that of the wild-
type. In contrast, its k

cat
and therefore the catalytic
efficiency was significantly lower (k
cat
13.85 ±
0.09 min
)1
, k
cat
/K
m
0.46 · 10
6
M
)1
Æs
)1
).
Biochemical characterization of S86C
Mutant S86A was inactive in the DTNB-HPLC assay
(Fig. 2A). Therefore, we decided to use other assays
developed in previous work. The removal of [1-
14
C]acetate
or [
14
C]proline from the corresponding acyl-S-Ppant-PCPs
is detected by the decrease in radioactivity covalently
attached to the protein fraction on addition of TEII
srf

[17].
AsshowninFig.2,intheabsenceofTEII,averyslow
ÔbackgroundÕ hydrolysis (decreasing enzyme-bound radio-
activity) occurred over the time scale observed. However,
the enzyme-bound radioactivity decreased rapidly on
addition of S86C to an assay mixture containing [1-
14
C]ace-
tyl-S-Ppant-PCP (Fig. 2B), confirming the enzyme’s func-
tionality. On the other hand, with [
14
C]Pro-S-Ppant-PCP
(Fig. 2C) as substrate, hydrolysis was only slightly above
background rates. Although the radioactive assay is not
suitable for absolute quantification of reaction rates, in the
case of the [
14
C]Pro-S-Ppant-PCP substrate it became
obvious that the enzymatic activity of S86C is reduced
compared with the wild-type enzyme [17].
In the case of a catalytic triad, one would expect a
reaction intermediate in which the acyl group is covalently
attached to the active-site Ser or Cys of the enzyme.
However, all attempts to detect such an enzyme-bound
intermediate with the S86C mutant by using the radioactive
assays in combination with SDS/PAGE analysis followed
by autoradiography of the gels failed. In no case was
thioesterase-bound radioactivity observed (data not shown).
Obviously, the reaction was still too fast to capture such an
intermediate.

Fig. 2. Biochemical characterization of TEII
srf
mutant S86C.
(A) DTNB-HPLC assay: acetyl-4¢-S-Ppant-PCP is used as a model
substrate for TEII
srf
[17]. The products formed are HS-4¢-S-Ppant-
PCP (holo-PCP) and acetic acid. Product analysis is performed with an
HPLC method, which requires modification of the free thiol of the HS-
holo-PCP with DTNB, resulting in TNB-holo-PCP. Therefore, DTNB
had to be added to the reaction mixture. TNB-holo-PCP, acetyl-holo-
PCP and apo-PCP can be separated from each other by an optimized
HPLC method [17]. S86C hydrolyses the substrate directly at the
beginning of the reaction very efficiently compared with the wild-type
enzyme. However, after less than 1 min, the enzyme becomes com-
pletely inactivated. This inhibition of S86C is due to the covalent
modification of the active-site Cys with TNB as judged by ESI-MS
experiments. (B) Radioactive assay: [1-
14
C]acetyl-4¢-S-Ppant-PCP is
used as substrate for TEII
srf
as previously described [17]. Therefore,
apo-PCP was converted into [1-
14
C]acetyl-4¢-S-Ppant-PCP by the
action of Sfp6 with [1-
14
C]acetyl-CoA as substrate. The assay mixture
was then divided; S86C was added to one part and omitted from the

other. Hydrolytic activity was observed in the absence of DTNB. The
enzyme-bound radioactivity decreased rapidly on addition of S86C.
Therefore, the catalytic efficiency of the mutant appeared to still be
very high. (C) Proline-quench assay: a recombinant NRPS module
(ProCAT) was allowed to activate and covalently load
14
C-labeled
proline. The assay mixture was then split; S86C was added to one part
and omitted from the other. The loss of radioactivity seems to be
slightly increased in the presence of S86C. However, the hydrolytic rate
is significantly decreased compared with previous results gained with
wild-type TEII
srf
[17].
Ó FEBS 2004 Mechanistic studies on TEII
srf
(Eur. J. Biochem. 271) 1541
Secondary-structure validation of the inactive mutants
by CD spectroscopy
CD spectroscopy is an easy and fast method to determine
the relative values of a-helices, b-sheets and loops within a
secondary structure of a protein, although it gives no
detailed structural information. To investigate the folding of
the inactive or unstable mutants S86A, D163A, D190A,
H216L and H216R, they were dialyzed against 50 m
M
sodium phosphate buffer and subjected to CD spectro-
scopy. Under these conditions, D163A and H216L preci-
pitated and could not be measured. This indicated that the
enzyme structures became unstable as a result of the

mutation of the strictly conserved Asp163 to Ala and
His216 to Leu. The spectra obtained for mutants S86A,
D190A and H216R looked very similar to that of wild-type
TEII
srf
and to each other (data not shown).
The computer-aided evaluation of the spectra resulted in
relative numbers for the secondary-structure elements,
which are presented in Table 4. These values are in the
same ranges for all four enzymes, indicating no significant
destruction of the enzyme structures caused by the intro-
duction of the mutations.
TNB modification of TEII
srf
and TEII
srf
mutants
DTNB, which was used as reagent in the DTNB-HPLC
assay for the determination of the kinetic parameters, reacts
with free thiol groups. As TEII
srf
contains three Cys
residues, of which Cys18 is highly conserved, and mutant
S86C showed no activity in the DTNB-HPLC assay, we
were interested to determine if, and how many, Cys residues
will be modified by the reagent. MS methods were used to
address this question. Wild-type TEII
srf
, C18A, S86A, and
S86C were incubated at room temperature in the presence

of DTNB for 30 min. For ÔquenchingÕ of the assays, the
reaction mixtures were purified very quickly on small gel
filtration columns. The DTNB-free enzyme fractions were
subsequently applied to ESI-TOF mass analysis, and the
number of TNB molecules covalently attached to the
proteins was determined.
The results of these modification studies are summarized
in Table 5. Wild-type TEII
srf
and S86A mutant were both
modified with one molecule of TNB. A portion of these
enzymes remained unmodified, indicating a slow reaction
with the reagent DTNB. In contrast, two TNB molecules
were exclusively observed to be covalently attached to
mutants C18A and S86C. For mutant S86C, the result
explained our biochemical data. Obviously one molecule of
TNB binds to the active-site Cys86 and thereby inactivates
the enzyme in the DTNB-HPLC assay. However, mutant
C18A showed the modification of both remaining Cys
residues, while the parent enzyme, containing three cyste-
ines, was modified by only one molecule of TNB in a slow
reaction. This leads to the conclusion that the mutant’s
structure may be changed to some extent. Therefore, the
small increase in K
m
observed for mutant C18A could be
due to this conformational change rather than to direct
involvement of this residue in substrate binding.
Discussion
For a long time, the biochemical role of TEIIs, which are

encoded by distinct genes associated with microbial NRPS
and PKS operons, was a matter of speculation. Recently,
however, biochemical studies have suggested a possible role
Table 4. Percentage distribution of secondary-structure elements in
wild-type TEII
srf
and the inactive mutants. Mutants D163A and H216L
were insoluble under the required buffer conditions.
TEII
srf
a-Helices b-Sheets Loops Undefined
wt 58.3 38.6 0.0 3.1
S86A 53.3 38.9 0.0 7.7
D163A – – – –
D190A 54.1 37.6 0.0 8.3
H216L – – – –
H216R 52.0 42.5 0.0 5.5
Table 5. ESI-TOF results of TEII
srf
or TEII
srf
mutant reaction with DTNB. The calculated values are without the starting methionine, which was
missing in all cases. wt, Wild-type; ND, not detected.
Unmodified 1 molecule TNB 2 molecules TNB
Calculated Measured Calculated Measured Calculated Measured
wt TEII
srf
28425.4 28424.7 28622.6 28623.1 28819.8 ND
C18A 28393.3 ND 28590.5 ND 28787.7 28788.1
S86A 28409.4 28408.8 28606.6 28607.7 28803.8 ND

S86C 28441.5 ND 28634.6 ND 28835.8 28834.1
Table 3. Summary of TEII
srf
and TEII
srf
mutant activities.
TEII
srf
K
m
(l
M
) k
cat
(min
)1
) k
cat
/K
m
(
M
)1
Æs
)1
)
Wild-type 0.9 ± 0.4 95 ± 5 1.75 · 10
6
C18A 1.7 ± 0.4 99 ± 4 0.97 · 10
6

H85A 0.5 ± 0.03 13.85 ± 0.09 0.46 · 10
6
S86C Active, but not suitable for HPLC-DTNB assay
S86A Inactive
S112A 0.2 ± 0.7 98 ± 10 8.2 · 10
6
D163A Active, but unstable during the HPLC-DTNB
assay
D189A 0.6 ± 1.2 154 ± 18 4.00 · 10
6
D190A Inactive
DD189/190AA Inactive
H216L Inactive
H216R Inactive
1542 U. Linne et al.(Eur. J. Biochem. 271) Ó FEBS 2004
for these TEIIs in the regeneration of mis-acylated NRPSs
and PKSs. They are obviously involved in removing short
acyl-S-Ppant intermediates from acyl carrier proteins and
PCPs associated with secondary metabolite biosynthesis
[15–17]. Enabled by the discovery of the natural substrate of
microbial secondary metabolism TEIIs [17], we set out to
determine the mechanistic properties of these enzymes [15–
17]. Therefore, the TEII
srf
associated with surfactin biosyn-
thesis in Bacillus subtilis was used as a prototype TEII for
our mechanistic studies. Because of the surprisingly high
identities (> 20%) between these TEII enzymes of micro-
bial secondary metabolism and a mammalian TEII (TEII
rat

[19]), which has been intensively studied [19,21–23,33,35,36],
the presence of a catalytic triad in microbial TEIIs was
postulated [24]. In TEII
rat
, Ser101, Asp236 and His237 were
reported to be involved in catalysis [21–23], although the
D236A mutant showed a residual activity of 40% [21].
There are two possible reasons for this: (a) Asp236 of
TEII
rat
is not part of the catalytic triad of this class of
hydrolases; (b) a catalytic diad (Ser-His) is sufficient for
catalytic activity. As our sequence alignment revealed that
Asp236 of TEII
rat
is not conserved in microbial TEIIs of
secondary metabolism, it was more likely that one residue of
the proposed catalytic triad had not been identified so far.
In agreement with the results for the mammalian TEII,
we confirmed that the corresponding residues Ser86 and
His216 are part of the catalytic triad in TEII
srf
of microbial
secondary metabolism. Mutants S86A and H216L showed
no hydrolytic activity. In addition, the S86C mutant was
inhibited by DTNB, and active in its absence. As judged
by ESI-TOF high-resolution MS, this inhibition was due to
covalent modification of the active-site Cys with one
molecule of TNB. Interestingly, H216R was also completely
inactive in the DTNB-HPLC assay. Therefore it seems that

Arg cannot functionally replace His216 in TEII
srf
.However,
inthecaseofTEII
rat
H237R, the residual activity reported
was reduced more than threefold compared with the parent
enzyme, which was only slightly above the detection limit
[23].
The second strictly conserved His residue found in TEII
srf
(His85) is located directly next to the active-site Ser86. The
observed reduction in catalytic efficiency may be caused by
repositioning of the Ser86 in mutant H85A resulting from
the replacement of His with Ala rather than by direct
involvement in catalysis.
To determine the identy of the remaining residue of the
proposed catalytic triad, the two strictly conserved Asp
residues among all TEIIs aligned (Asp163 and Asp190 in
TEII
srf
) were separately exchanged with Ala (D163A and
D190A mutants). Our results clearly indicate that Asp190
(Asp212 in TEII
rat
) is the missing member of the catalytic
triad, whereas Asp163 (Asp183 in TEII
rat
) seems to be
structurally important, as evidenced by the precipitation of

the enzyme when dialyzed against phosphate buffer and the
observed instability when assayed at 37 °C for several
minutes or after storage at )80 °C. In contrast, wild-type
TEII
srf
and the other mutants studied showed no instability
under these conditions.
Many such hydrolases that have a catalytic triad belong
to the large class of a/b-hydrolases. They show a conserved
characteristic fold, which was first described by Ollis et al.
[37]. This fold was also recently reported for the thio-
esterases of type I, which are located as internal domains
at the C-termini of the termination modules of NRPS-
biosynthetic and PKS-biosynthetic enzymes [31,38]. For the
latter type I thioesterases, a catalytic triad was biochemically
confirmed in the case of the TE domain of surfactin
synthetase C [11].
The canonical a/b-hydrolase fold, which is illustrated
in Fig. 3, consists of eight b-strands (1–8), which are
positioned in plane. Above them are positioned two (A and
F) and under them four (B, C, D, and E) a-helices. The
nucleophile of the catalytic triad (Ser86 in TEII
srf
)isalways
located at the Ônucleophile elbowÕ in a G-x-Nu-x-G core
sequence (x, any amino acid; Nu, nucleophile). The
nucleophile elbow is a loop directly after b-strand 5. The
acidic residue of the triad, an Asp residue, is located on a
loop following b-strand seven (Asp190 in TEII
srf

). Finally,
the catalytic triad is completed by the His residue, which is
located on a longer loop between b-strand 8 and a-helix F
(His216 in TEII
srf
). Based on the relative positioning and
distances between the three residues forming the catalytic
triadinTEII
srf
(Ser86, Asp190, and His216) as well as the
existence of the core motif ÔGHSxGÕ, which is always found
in a/b-hydrolases (G-x-Nu-x-G, see above), there is strong
evidence that the microbial TEIIs of secondary metabolism
as well as the TEII
rat
belong to this large class of enzymes,
too. However, no structural data are available on them.
In summary, we have clearly identified a catalytic triad in
the prototype TEII
srf
consisting of Ser86, His216 and
Asp190. Moreover, because of the remarkably high simi-
larities of the microbial TEIIs of secondary metabolism to
Fig. 3. Schematic representation of the canonical a/b-hydrolase fold
[50]. (A) Three-dimensional structure. (B) Two-dimensional repre-
sentation.
Ó FEBS 2004 Mechanistic studies on TEII
srf
(Eur. J. Biochem. 271) 1543
the mammalian TEII

rat
, our results strongly suggest that
Asp212 is the acidic residue of the proposed catalytic triad in
TEII
rat
. With this knowledge, the reduction in catalytic
efficiency of the TEII
rat
mutant D236A, which was observed
by Tai et al. [21] is probably more likely to be due to the
mutation directly adjacent to the catalytic His237 than to a
direct involvement in catalysis. Furthermore, the relative
positioning of the residues of the catalytic triad in TEII
srf
and TEII
rat
provides evidence that this class of enzymes
belongs to the large family of a/b-hydrolases.
Acknowledgements
We thank Antje Scha
¨
fer for excellent technical assistance and protein
purification. The CD spectroscopy was carried out in the Laboratory of
Professor T. Carell with the assistance of Alexandra Mees. We also
thank Mohammad R. Mofid for providing CD spectroscopy data on
wild-type TEII
srf
. This work was funded by the Deutsche Forschung-
sgemeinschaft and the Fonds der chemischen Industrie.
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