Marahiel Philipps Universita¨t Marburg, Fachbereich Chemie/Biochemie, Germany Recent studies on type II thioesterases TEIIs involved in microbial secondary metabolism described a role fo
Trang 1Mutational 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 (TEIIsrf), 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 TEIIsrf were constructed and biochemically
investigated We clearly identified a catalytic triad consisting
of Ser86, Asp190 and His216, suggesting that TEIIsrfbelongs
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 eukaryprokary-otes, 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: marahiel@chemie.uni-marburg.de
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 (one letter–one domain 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)
Trang 2the 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 showremarkably high sequence similarities
(> 20%) and length ( 240–260 residues) to a specialized
mammalian (rat) TEII (TEIIrat), 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
TEIIrat, 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 TEIISrf (242
amino acids, 28 kDa), which is associated with the
production of the secondary metabolite surfactin [12,24]
A total of 11 TEIISrfmutants, 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 TEIIratof 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 (http://www.ncbi.nih.gov/)
Sequences used were derived from NRPS and PKS
biosynthetic clusters, in addition to that of the mammalian TEIIrat The sequences ( 250 amino-acid stretches) were aligned using the programMEGALIGNfrom the DNA Star package, applying the method of Jotun Hein with default parameters
Construction of TEIIsrfand TEIIsrfmutant expression plasmids and protein purification
Cloning and overproduction of wild-type TEIIsrfhas been described previously [17] All mutants generated in this study were introduced into plasmid pTEIIsrf[17] Mutants were obtained using the Quick Change Site Directed Mutagenesis KitTM(Stratagene, Heidelberg, Germany) as described by manufacturer The mutants constructed and the primers used with plasmid pTEIIsrf 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 coliM15/pREP4 was transformed separately with all mutant plasmids constructed Expression and purification of the His6-tagged enzymes (except D163A) were performed as previously described for wild-type TEIIsrf [17] In the case of D163A, an overnight culture of the expression strain was grown at 22C and induced with iso-propyl thio-b-D-galactoside (0.2 mM) After an additional
3 h incubation at 22C, the cells were centrifuged at 4 C, and D163A was then purified, dialyzed against assay buffer using HiTrapTMDesalting 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 Ni2+-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).
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 GC T 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
3¢ GG CGG CTG GAT TGC AGC AAT GAT AAC CGC C
3¢ C AAG AGC CCG GTA AGC TGA TCG GAA AGA AGG C
3¢ C CGC ATC TCG TAT GCA TTT TTT ATC AGC AAG CCC GTT AAA 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
3¢ GA CAG CAG GAA CAT GAG CCC GCC GTC AAA TTG
Trang 3using UV spectroscopy (A280) The calculated absorption
coefficients of wild-type TEIIsrfand TEIIsrfmutants are very
similar The value of the wild-type TEIIsrf(16 860M )1Æcm)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 TEIIsrf
-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 TEIIsrfand TEIIsrfmutants, was carried out
according to previously developed protocols [17]
5,5¢-Dithiobis(2-nitrobenzoic acid) (DTNB)-HPLC assay
TEIIsrfor TEIIsrfmutants at a concentration of 25 nMwere
incubated at 37C with various concentrations of
acetyl-S-Ppant-PCP (2–35 lMin the case of active mutants; initial
concentration for activity test of all mutants was 25 lM) in
the presence of DTNB (8 lL of a 10 mMstock solution) in
assay buffer (50 mMHepes, 100 mMNaCl, 1 mMEDTA,
10 mM MgCl2, pH 7.0) in a total volume of 400 lL
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 [1-14C]acetyl-S-Ppant-enzyme hydrolysis
Apo-PCP [17] was incubated at a concentration of 1 mM
in a total volume of 1.3 mL with 20 lM[1-14C]acetyl-CoA
(50 mCiÆmmol)1; ICN, Eschw ege, Germany), 50 nM Sfp,
and 10 mMMgCl2in assay buffer (50 mMHepes, 100 mM
NaCl, 1 mM EDTA, pH 7.3) at 37C After complete
modification, the reaction mixture was split and TEIIsrf
or TEIIsrf mutants were added to one aliquot to a final
concentration of 500 nM in the case of PCP Samples
(100 lL) were taken at defined time points and mixed with
800 lL trichloroacetic acid (10%, w/v) and 15 lL BSA
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 lMin a total volume of 1.5 mL with 10 mMMgCl2, 5 mM
ATP and 4.1 lM [14C]Pro (246 mCiÆmmol)1; Hartmann
Analytics, Braunschweig, Germany) in assay buffer at
37C After complete aminoacylation, 15 lL of a 100 mM
unlabeled proline solution was added, and the reaction
mixture was split TEIIsrfor TEIIsrfmutants were added to
one aliquot to a final concentration of 500 nM At defined
time points, 100 lL samples were removed and prepared
and analyzed as described above
TNB modification of TEIIsrfand TEIIsrfmutants DTNB (4 lL of a 10 mMsolution in dimethyl sulfoxide) was added to 46 lL of a 25 lM solution of TEIIsrf, TEIIsrfC18A, TEIIsrfS86C or TEIIsrfS86A and incubated for 30 min at room temperature Subsequently, the yellow reaction mixture was applied to MicroSpinTM 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 suppliedANALYSTTMsoftware
CD spectroscopy of TEIIsrfmutants For CD spectroscopy, TEIIsrf and TEIIsrf mutants were dialyzed against 50 mM sodium phosphate buffer (pH 7; 19.5 mM NaH2PO4 and 30.5 mM Na2HPO4) and diluted
in the same buffer to a final concentration of 10 lM 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 25C 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 TEIIsrf
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 TEIIrat were aligned A catalytic role for Ser101, Asp236 and His237 in TEIIrat has been reported [21] The relative identity scores between TEIIsrfand the other TEIIs range from 20.3% (MegH [27] and TEIIrat[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 Interestingly, the overall identity
Trang 4compared 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 TEIIrat, w hich w as
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
TEIIrat, 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 reacdetec-tion intermediates [22] His216, which is
the corresponding residue in TEIIsrfto the active-site His237
of TEIIrat [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 TEIIratwith 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 TEIIsrfand four in TEIIrat, whereby only the one mutated (Cys18 in TEIIsrf) is conserved Furthermore, the assay used for the kinetic characterization
of TEIIsrfdepends 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 TEIIsrf mutants
We constructed a set of 10 single TEIIsrfmutants (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 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].
MegH
TeII rat
Trang 5Expression and purification was carried out as described
previously for wild-type TEIIsrf[17] Because D163A was
unstable when expressed at 30C under standard
condi-tions and precipitated when dialyzed against 50 mMsodium
phosphate buffer (the others did not; see the paragraph
about CD spectroscopy of the TEIIsrf mutants), the
expression was performed at a lower temperature (22C)
As judged by SDS/PAGE, all His6-tagged recombinant
proteins were purified to near-homogeneity by single-step
Ni2+-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
TEIIsrf, which is hydrolyzed very efficiently by the enzyme
(Km¼ 0.9 ± 0.4 lM; kcat¼ 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 lM, which is significantly higher
than the Km of TEIIsrf (0.9 ± 0.4 lM [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 fewdays at )80 C Furthermore, as mentioned above, it precipitated during dialysis against sodium phosphate buffer Therefore, the enzyme was expressed at a lower temperature (22C), 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 37C, no further hydrolysis of the remain-ing substrate was observed, indicatremain-ing 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 Km values obtained for C18A (1.7 ± 0.4 lM), S112A (0.2 ± 0.7 lM) and D189A (0.6 ± 1.2 lM) were in the same range as the wild-type Km(0.9 ± 0.4 lM[17]) The same was true for
kcat(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 kcat/Km (C18A 0.97· 106M )1Æs)1, S112A 8.2· 106M )1Æs)1, D189A 4· 106M )1Æs)1, and wild-type 1.75· 106M )1Æs)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).
Trang 6and D189A) had only a minor effect on the catalytic
efficiency of the enzyme The Km of mutant H85A
(0.5 ± 0.03 lM) was also very close to that of the
wild-type In contrast, its kcat and therefore the catalytic
efficiency was significantly lower (kcat 13.85 ±
0.09 min)1, kcat/Km0.46· 106
M )1Æs)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 [1-14C]acetate
or [14C]proline from the corresponding acyl-S-Ppant-PCPs
is detected by the decrease in radioactivity covalently
attached to the protein fraction on addition of TEIIsrf
[17]
As show n in Fig 2, in the absence of TEII, a very slow
background 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 [14C]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 [14C]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-HS-4¢-S-Ppant-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-HS-holo-PCP Therefore, DTNB
had to be added to the reaction mixture TNB-holo-PCP,
acetyl-holo-PCP and apo-acetyl-holo-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: [1-14C]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 [1-14C]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 load14C-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].
Trang 7Secondary-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 mM
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
TEIIsrfand 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 TEIIsrfand TEIIsrfmutants DTNB, which was used as reagent in the DTNB-HPLC assay for the determination of the kinetic parameters, reacts with free thiol groups As TEIIsrf 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 TEIIsrf, C18A, S86A, and S86C were incubated at room temperature in the presence
of DTNB for 30 min For quenching 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 TEIIsrfand S86A mutant were both modified with one molecule of TNB A portion of these enzymes remained unmodified, indicating a slowreaction 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 Kmobserved 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.
Table 5 ESI-TOFresults 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.
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
H85A 0.5 ± 0.03 13.85 ± 0.09 0.46 · 10 6
S86C Active, but not suitable for HPLC-DTNB assay
D163A Active, but unstable during the HPLC-DTNB
assay
DD189/190AA Inactive
Trang 8for 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 TEIIsrfassociated 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 (TEIIrat
[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 TEIIrat, 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
TEIIrat 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 TEIIrat 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 TEIIsrfof 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 TEIIsrf How ever,
in the case of TEIIratH237R, 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 TEIIsrf
(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
TEIIsrf) were separately exchanged with Ala (D163A and
D190A mutants) Our results clearly indicate that Asp190
(Asp212 in TEIIrat) is the missing member of the catalytic
triad, whereas Asp163 (Asp183 in TEIIrat) 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 37C for several
minutes or after storage at)80 C In contrast, wild-type
TEIIsrfand 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 showa 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-NRPS-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 TEIIsrf) is alw ays located at the nucleophile elbow in a G-x-Nu-x-G core sequence (x, any amino acid; Nu, nucleophile) The nucleophile elbowis 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 TEIIsrf) 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 TEIIsrf) Based on the relative positioning and distances between the three residues forming the catalytic triad in TEIIsrf(Ser86, Asp190, and His216) as well as the existence of the core motif GHSxG, 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 TEIIratbelong 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 TEIIsrf 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.
Trang 9the mammalian TEIIrat, our results strongly suggest that
Asp212 is the acidic residue of the proposed catalytic triad in
TEIIrat With this knowledge, the reduction in catalytic
efficiency of the TEIIratmutant 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 TEIIsrf
and TEIIrat 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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