Structural and biochemical characterization of the salicylyl-acyltranferase SsfX3 from a tetracycline biosynthetic pathway

Article abstract of DOI:10.1074/jbc.M111.299859

SsfX3 is a GDSL family acyltransferase that transfers salicylate to the C-4 hydroxyl of a tetracycline intermediate in the penultimate step during biosynthesis of the anticancer natural product SF2575. The C-4 salicylate takes the place of the more common C-4 dimethylamine functionality, making SsfX3 the first acyltransferase identified to act on a tetracycline substrate. The crystal structure of SsfX3 was determined at 2.5A , revealing two distinct domains as follows: an N-terminal β-sandwich domain that resembles a carbohydrate- binding module, and a C-terminal catalytic domain that contains the atypical α/β-hydrolase fold found in the GDSL hydrolase family of enzymes. The active site lies at one end of a large open binding pocket, which is spatially defined by structural elements from both the N- and C-terminal domains. Mutational analysis in the putative substrate binding pocket identified residues from both domains that are important for binding the acyl donor and acceptor. Furthermore, removal of the N-terminal carbohydrate-binding module- like domain rendered the stand-alone α/β-hydrolase domain inactive. The additional noncatalytic module is therefore proposed to be required to define the binding pocket and provide sufficient interactions with the spatially extended tetracyclic substrate. SsfX3 was also demonstrated to accept a variety of non-native acyl groups. This relaxed substrate specificity toward the acyl donor allowed the chemoenzymatic biosynthesis of C-4-modified analogs of the immediate precursor to the bioactive SF2575; these were used to assay the structure activity relationships at the C-4 position.

Full text of DOI:10.1074/jbc.M111.299859

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 48, pp. 41539–41551, December 2, 2011
Printed in the U.S.A.
Structural and Biochemical Characterization of the
Salicylyl-acyltranferase SsfX3 from a Tetracycline
ꢀ
□
S
Biosynthetic Pathway*
Received for publication, September 1, 2011, and in revised form, September 28, 2011 Published, JBC Papers in Press, September 30, 2011, DOI 10.1074/jbc.M111.299859
Lauren B. Pickens‡, Michael R. Sawaya§¶, Huma Rasool‡, Inna Pashkov§, Todd O. Yeates§ʈ, and Yi Tang‡§ʈ1
From the Departments of ‡Chemical and Biomolecular Engineering and §Chemistry and Biochemistry, ʈMolecular Biology Institute,
¶Howard Hughes Medical Institute, and UCLA-DOE Institute for Genomics and Proteomics, UCLA, Los Angeles, California 90095
Background: SsfX3 is an acyltransferase that can acylate tetracycline-like molecules at C-4.
Results: Crystal structure showed SsfX3 contains a structural N-terminal ␤-sandwich domain and a catalytic C-terminal
hydrolase domain. Mutagenesis revealed both are essential for activity.
Conclusion: The N-terminal domain is recruited to bind the large “small molecule” substrate.
Significance: New structural knowledge enables engineering of the acyltransferase toward synthesis of tetracycline analogs.
SsfX3 is a GDSL family acyltransferase that transfers salic-
ylate to the C-4 hydroxyl of a tetracycline intermediate in the
penultimate step during biosynthesis of the anticancer natu-
ral product SF2575. The C-4 salicylate takes the place of the
more common C-4 dimethylamine functionality, making
SsfX3 the first acyltransferase identified to act on a tetracy-
cline substrate. The crystal structure of SsfX3 was deter-
Natural products display an incredibly large spectrum of
chemical diversity, in large part due to the many combinations
of tailoring enzymes that decorate their scaffolds. Group trans-
ferases such as glycosyltransferases, methyltransferases, acyl-
transferases, aminotransferases, and prenyltransferases play
key roles in the biosynthesis of many important natural prod-
ucts, such as doxorubicin and erythromycin, and have been
successfully used for the combinatorial biosynthesis of unnat-
ural analogs (1). The acyl transfer modification has in many
cases been shown to be vital to arming the final products with
bioactivity, as seen with the biosynthesis of teicoplanin (2, 3),
chromomycin (4), phoslactomycin (5), and lovastatin (6). Acyl-
transferases frequently show broad substrate flexibility with
regard to the donor acyl group, and therefore they can be valu-
able tools to diversify natural product scaffolds at positions
important for bioactivity.
˚
mined at 2.5 A, revealing two distinct domains as follows: an
N-terminal ␤-sandwich domain that resembles a carbohy-
drate-binding module, and a C-terminal catalytic domain
that contains the atypical ␣/␤-hydrolase fold found in the
GDSL hydrolase family of enzymes. The active site lies at one
end of a large open binding pocket, which is spatially defined
by structural elements from both the N- and C-terminal
domains. Mutational analysis in the putative substrate bind-
ing pocket identified residues from both domains that are
important for binding the acyl donor and acceptor. Further-
more, removal of the N-terminal carbohydrate-binding mod-
ule-like domain rendered the stand-alone ␣/␤-hydrolase
domain inactive. The additional noncatalytic module is
therefore proposed to be required to define the binding
pocket and provide sufficient interactions with the spatially
extended tetracyclic substrate. SsfX3 was also demonstrated
to accept a variety of non-native acyl groups. This relaxed
substrate specificity toward the acyl donor allowed the che-
moenzymatic biosynthesis of C-4-modified analogs of the
immediate precursor to the bioactive SF2575; these were
used to assay the structure activity relationships at the C-4
position.
SF2575 is a tetracycline natural product produced by Strep-
tomyces sp. SF2575. It has potent anticancer activity (7, 8).
SF2575 is structurally unique among tetracyclines due to its
extensive tailoring modifications, including glycosylation with
D-olivose at C-9, acylation of the C-4 hydroxyl with salicylate,
and acylation of the C-4Ј of D-olivose with angelate. The ssf gene
cluster encoding these activities was identified recently, ena-
bling the biosynthetic pathway for SF2575 to be articulated (Fig.
1) (9). Transfer of the salicylyl and the angelicyl groups to the
aglycon of SF2575 are both important for the anticancer activ-
ity of SF2575 (8). Understanding the enzymatic basis for these
pendant group additions offers the possibility of structure-ac-
tivity relationship studies and subsequent fine-tuning of the
pharmacological properties of SF2575. Toward this end, we
previously identified SsfX3 as the acyltransferase responsible
for transfer of salicylate from salicylyl-CoA 5 to the C-4
hydroxyl of the key intermediate 4 to produce the C-4-(R)-
salicylated 6 (Fig. 1) (9). The salicylyl moiety transforms the
inactive 4 into the moderately potent cytotoxic compound 6.
* This work was supported by National Science Foundation Chemical, Bioen-
gineering, Environmental, and Transport Systems (CBET) Grants 0545860
and 1033070 (to Y. T.).
ꢀ This article was selected as a Paper of the Week.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1 and Fig. S1.
Theatomiccoordinatesandstructurefactors(code3SKV)havebeendepositedin Engineered biosynthesis of new tetracycline compounds has
the Protein Data Bank, Research Collaboratory for Structural Bioinformatics,
been limited in part by the dearth of specific modifying
Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
1 To whom correspondence should be addressed: UCLA, Los Angeles, CA
90095. E-mail: yitang@ucla.edu.
enzymes. SsfX3 therefore represents a possible tool to specifi-
cally decorate the C-4 position of the tetracycline aglycon. This
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Tetracycline Acyltransferase SsfX3
FIGURE1. Abbreviated biosyntheticpathwayofSF2575showing key intermediates and tailoring steps. The upstreamsequence of reactions isproposed
based on the structures of intermediates 1–3.
prompted us to further study the structural and mechanistic and co-workers (24) tested the activities of several known
features of this enzyme.
GDSL hydrolases toward a variety of esterase, lipase, thioes-
terase, phospholipase, and protease substrates and found
each of the hydrolases tested to have a unique activity profile
for a variety of substrates. Huang and co-workers (25, 26)
performed NMR spectroscopy on the TAP enzyme and
showed that the active site is highly flexible, which could
contribute to the broad substrate specificity.
A protein family database search revealed that the C-termi-
nal domain of SsfX3 has low but recognizable sequence simi-
larity to the GDSL hydrolase family of enzymes (with an E value
of 0.071). This family was first suggested by Upton and Buckley
(10), who identified a conserved active site motif at the N ter-
minus of the enzyme (rather than the middle as observed for
typical ␣/␤-hydrolases), as well as identifying five conserved
sequence blocks. This family of enzymes contains the con-
served GDSL(S) motif around the active serine, lacks a well
defined nucleophilic elbow or sharp turn identified by the
canonical GXSXG motif (11), and has four invariantly con-
served residues making up the oxyanion hole, Ser, Gly, Asn, and
His; these features allowed the additional protein to be classi-
fied as members of the SGNH hydrolase family of enzymes (12).
One of the best characterized GDSL hydrolases is Escherichia
coli thioesterase I (TAP),2 a versatile enzyme known to function
as a thioesterase, esterase, arylesterase, protease, and lysophos-
pholipase (13). Its promiscuity led TAP to be identified as three
different proteins, thioesterase I (TesA) (14), protease I (ApeI)
(15), and lysophospholipase L1 (PlcC) (16) before Nojima and
co-workers (17) found that they were identical molecules
encoded by the same gene. The protein family database cur-
rently identifies 6817 sequences belonging to the GDSL
hydrolase family (18). Several of these have been structurally
characterized, including E. coli TAP (13, 19, 20), Streptomy-
ces scabies SsEst (21), rhamnogalacturonan acetylesterase
from Aspergillus aculeatus (22), and esterase EstA from
Pseudomonas aeruginosa (23). GDSL hydrolases are also
observed to be promiscuous with regard to substrate. Asler
The GDSL hydrolase consensus sequence mapped only
to the C-terminal half of the SsfX3 protein, leaving the function
of the N-terminal half unknown. A number of homologs of
SsfX3 were identified based on amino acid sequence similarity
using BLAST, most of them with uncharacterized functions.
Only four SsfX3 homologs from verified natural product gene
clusters have been identified to date. The closest homolog in
the NCBI database is AviX9, a putative GDSL hydrolase with
unknown function from the avilamycin gene cluster (27).
Recently, GDSL hydrolases have also been identified in the
caprazamycin (28), related liposidomycin (29), and A-90289
(30) gene clusters. These enzymes have been proposed to be
involved in the transfer of long chain fatty acyl groups to form
liponucleoside antibiotics. Sequence similarity of these acyl-
transferases to SsfX3 is evident across both the novel N-termi-
nal and the GDSL-like C-terminal domains. To gain further
insight into the role of these enzymes as atypical acyltrans-
ferases in natural product biosynthesis, we determined the
x-ray crystal structure of SsfX3 and performed structurally
guided mutagenesis to probe key residues around the active site
and to identify residues important for catalysis and substrate
binding. The three-dimensional structure confirmed that
SsfX3 is indeed composed of two distinct domains separated by
a long linker. The N-terminal domain bears resemblance to a
carbohydrate-binding module (CBM) domain and is critical for
substrate binding, whereas the catalytic C-terminal domain
2 The abbreviations used are: TAP, E. coli thioesterase I; CBM, carbohydrate-
binding module; MAD, multiwavelength anomalous dispersion; r.m.s.d.,
root mean square deviation; PDB, Protein Data Bank.
41540 JOURNAL OF BIOLOGICAL CHEMISTRY
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Tetracycline Acyltransferase SsfX3
displays the ␣/␤-hydrolase fold typical of the GDSL hydrolase mutant protein migrated as a single band on SDS-PAGE and
family.
subsequently afforded the highest quality crystals for x-ray data
collection. Kinetic assays verified that the mutation had no
impact on the enzyme function. Protein crystals were grown by
EXPERIMENTAL PROCEDURES
SsfX3 and Mutant Plasmid Construction—The gene encod- the hanging drop vapor diffusion method at room temperature.
ing SsfX3 was amplified by PCR from cosmid 5F15 (9) and Native crystals were grown from a reservoir containing 3.3 M
ligated to pCR-Blunt vector (Invitrogen) to generate plasmid sodium formate, pH 7.0, with an additive solution giving a final
pLP59. The gene was excised from pCR-Blunt and cloned into concentration of 0.01 M reduced L-glutathione and 0.01 M oxi-
pET28a (Invitrogen) using NdeI and EcoRI restriction sites. dized L-glutathione in the drop. Selenomethionine crystals
SsfX3 mutants were prepared using pLP59 as a template. Full- were grown from a reservoir of 3.3 M sodium formate, pH 7, and
length PCR products were ligated into pCR-Blunt vector and a drop containing 2.0 M NDSB-201 (3-(1-pyridino)-1-propane
then excised using NdeI and EcoRI and ligated into pET28a. All sulfonate) from the Hampton Research additive screen. Crystal
genes were confirmed by DNA sequencing (Laragen) prior to trays were set up with a 1.0-␮l drop volume by a TTP Labtech
cloning into pET28a. Primers used for amplification and splic- Mosquito robot. The crystals appeared as rectangular plates
ing by overlap extension are given in supplemental Table S1. with the longest dimension less than 80 ␮m. The crystals
Expression plasmids were then transformed into E. coli required approximately 2 weeks to reach their full size.
BL21(DE3) for protein expression and purification.
Data Collection—X-ray diffraction data from the native crys-
Heterologous Expression and Purification of Enzymes— tal were collected at the Advanced Light Source, beamline 8.2.1,
SsfX3, SsfL1, and SsfX3 mutants used for kinetic assays and using an ADSC Quantum 315 3X3 CCD array. The crystal was
substrate preparation were expressed from E. coli BL21(DE3) as cooled to 100 K in a cryogenic nitrogen stream for data collec-
fusion proteins with an N-terminal hexahistidine tag. The tion. No additional cryo-protectant was included. Each 5-s
C-terminal domain of SsfX3 was expressed from pET24 as a exposure covered 1° of crystal rotation. The incident beam was
fusion protein with a C-terminal hexahistidine tag. E. coli not attenuated. A total of 120 images was used in the reported
BL21(DE3) transformants containing the plasmid of interest data set, which extended to 2.5 Å resolution (Table 1).
were used to inoculate a 5-ml culture containing LB medium
Diffraction data from two selenomethionine derivatives were
supplemented with 35 mg/liter kanamycin. The seed culture collected at the Advanced Photon Source, beamline 24-ID-C,
was grown overnight at 37 °C and added to a fresh 2-liter flask using the same model detector. Both selenomethionine deriva-
containing 500 ml of LB medium with 35 mg/liter kanamycin tive crystals were cryo-protected by 10-s swipes through a solu-
and grown at 37 °C until the A600 reached 0.5 to 0.8. The culture tion containing 7.0 ␮l of reservoir solution and 3.0 ␮l of 100%
was then induced with isopropyl 1-thio-␤-D-galactopyranoside glycerol and then cooled to 100 K in a cryogenic nitrogen
to a final concentration of 120 ␮M, and protein expression pro- stream for data collection. Each exposure was collected for 1 s
ceeded at 16 °C for 6 h. Selenomethioninyl SsfX3 was expressed and covered 1° of crystal rotation. Data from the first selenome-
by the inhibition of the methionine biosynthetic pathway in thionine crystal were collected using a 70-␮m diameter aper-
minimal media supplemented with selenomethionine as ture and 5% transmission. Data from the second selenomethio-
described by Doublie (31).
nine crystal were collected using a 30-␮m aperture and 10%
Protein purification was carried out at 4 °C. The cell pellets transmission. A total of 100 images was used for the data set at
were harvested by centrifugation and resuspended in Buffer A the selenium anomalous inflection wavelength, and 82 images
(50 mM Tris-HCl, pH 7.9, 10 mM imidazole, and 50 mM NaCl). were used in the high remote wavelength data set. The inverse
Cell membranes were disrupted by sonication. The cell lysate beam mode was not used for either data set. Data reduction and
was centrifuged at 16,000 rpm, and the soluble fraction was scaling for all crystals were performed using DENZO/SCALE-
collected and incubated with nickel-nitrilotriacetic acid resin PACK (33). Data collection statistics are reported in Table 1.
(GE Healthcare) for 30 min with gentle rotation. Protein/resin
Structure Determination and Refinement—In a multiwave-
mixture was added to a gravity flow column, and buffers con- length anomalous dispersion (MAD) experiment, three data
taining increasing concentrations of imidazole were applied sets at three different wavelengths were customarily collected
stepwise. The target proteins were eluted in Buffer A contain- from a single crystal to ensure isomorphism. However, because
ing 250 mM imidazole. An Amicon filtration column (Milli- of their small sizes, none of the crystals could withstand the
pore) was used for buffer exchange and concentration of the radiation-induced damage caused by a three-wavelength
protein solution. Purified enzymes were stored in Buffer B (50 experiment. An attempt to make do with a single wave-
mM Tris-HCl, pH 7.9, 2 mM EDTA, 2 mM DTT) with 10% glyc- length data set by calculating phases using the single wave-
erol for cryopreservation. Proteins were aliquoted at 0.5 mM length anomalous dispersion method did not result in an
concentration, flash-frozen on dry ice, and stored at Ϫ80 °C. interpretable electron density map. Instead, a MAD data set
Protein concentrations were measured by a Bradford assay was assembled from two crystals as follows: a data set col-
using bovine serum albumin (BSA) as a standard (32).
lected at the peak wavelength using one crystal, in combina-
Crystallization of SsfX3—Purified SsfX3 migrated as two tion with data collected at the inflection, and high remote
bands when loaded onto an SDS-polyacrylamide gel, possibly wavelengths of a second crystal (Table 1). The two selenome-
due to modification of the surface cysteine. To facilitate crys- thionine crystals were sufficiently isomorphous as indicated by
tallization, the nonconserved Cys-68, which resides in the the correlation coefficients between signed anomalous differ-
N-terminal domain, was mutated to histidine. The C68H ences reported by SHELXC in the program HKL2MAP (34).
DECEMBER 2, 2011•VOLUME 286•NUMBER 48
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Tetracycline Acyltransferase SsfX3
The correlation ranged between 0.55 and 0.25 over a resolution 300 GL) (GE Healthcare) at 4 °C using a running buffer consist-
range from 20.0 to 3.5 Å. Coordinates for 7 of 14 possible sele-
nium sites were obtained by SHELXD using both dispersive and
anomalous differences from the three data sets (35).
ing of 50 mM sodium phosphate, pH 7.0, and 150 mM NaCl.
Preparation of Tetracycline Substrates—Compound 4 was
obtained by incubation of a 10 mg/ml solution of SF2575 in
1.0 M NaOH for 15 h. To isolate compound 4, the reaction
mixture was adjusted to pH 7 by addition of 6.2 M HCl, and the
products were extracted with ethyl acetate/acetic acid (99:1%).
Following evaporation of the solvent, the product was redis-
solved in methanol and purified by HPLC (Alltech Altima
reverse phase column 5 ␮m, 10 ϫ 250 mm) with an isocratic
gradient of 78% CH3CN in water (0.1% TFA). SF2575 interme-
diates 1 and 2 were generously provided by Peng Wang
(UCLA).
Phases were calculated using MLPHARE and modified by
solvent flattening and histogram matching using DM from the
CCP4 suite of programs (36). The inflection data set was used as
the reference wavelength. Three additional selenium sites
could be identified in self-anomalous difference Fourier maps.
These were included in another round of phasing. The resulting
map was of sufficient quality to allow the manual placement of
several ␤-strands and ␣-helices with the program COOT (37).
At this point, it was possible to recognize a structural similarity
with the carbohydrate esterase from Clostridium thermocellum
(PDB code 2WAB) (38). Swiss-Model (39) had suggested its use
as the basis for building a homology model of SsfX3 before
phases could be obtained. Coordinates of PDB code 2WAB had
been used in attempts to solve the structure by the molecular
replacement method but without success. In retrospect, the
failure was probably due to the low sequence identity between
SsfX3 and 2WAB (less than 16%). With newly acquired phases,
two molecules of 2WAB could be placed in the asymmetric unit
using the phased rotation and translation function employed in
the program MOLREP (40). Phases were further improved by
2-fold symmetry averaging in DM using the coordinates of the
properly placed 2WAB molecules to construct a molecular
mask using the program MAMA (41). The initial NCS matrix
was obtained by least squares superpositioning of NCS-related
selenium atom coordinates using the program O (42) and then
improved using Kleywegt’s program IMP (43). Statistics for
experimental phases calculated to 2.9 Å are reported in Table 1.
The model for SsfX3 was initially refined with the PHENIX
(44)-simulated annealing algorithm and with REFMAC5 using
tight 2-fold noncrystallographic symmetry restraints (45).
Later, TLS parameterization of domain disorder was employed
(46). After each refinement step, the model was visually
inspected in Coot, using both 2Fo Ϫ Fc and Fo Ϫ Fc difference
maps. All hydrogen atoms connected to carbon atoms and
backbone nitrogen atoms were included at their geometrically
calculated positions and refined using a riding model. Finally,
the model was refined using Buster (47).
Preparation of Aryl-CoA Substrates—Aryl-CoA substrates
were prepared enzymatically using salicylyl-CoA ligase SsfL1.
Reactions were set up and contain the following: 50 mM HEPES,
pH 7.9, 10 mM MgCl2, 5 mM coenzyme-A, 6 mM acid substrates,
5 mM ATP, and 20 ␮M SsfL1. Reactions were incubated for at
least 2 h at room temperature. The reaction mix was then fil-
tered to remove precipitated protein and directly purified by
HPLC (Alltech Altima reverse phase column 5 ␮m, 10 ϫ 250
mm) with a gradient of CH3CN in water (0.1% TFA). The gra-
dient varied depending on the reaction as some products were
more hydrophilic than others, requiring adjustment to the
method. Butyryl-CoA lithium salt was purchased from Sigma
and used directly without further purification.
Kinetic Assays—The assays were performed at room temper-
ature in 50 mM HEPES, pH 7.9, with 10 mM MgCl2. To deter-
mine the KM value of compound 4, the concentration of 4 was
varied from 5 to 300 ␮M keeping concentration of compound 5
constant at 300 ␮M or at least 8-fold higher than observed KM
value for compound 5. To determine the KM value of com-
pound 5, the concentration of 5 was varied from 5 ␮M to 2 mM
keeping the concentration of compound 4 constant at 50 ␮M or
10-fold higher than observed KM value for compound 4. Con-
centration of SsfX3 was at 10 nM for all reactions. Concentra-
tions of SsfX3 mutants were optimized for each to be between
10 and 100 nM depending on the rate of reaction. A 1.2-ml
reaction volume was set up, and aliquots of 240 ␮l were
removed and quenched by extraction with 300 ␮l of ethyl ace-
tate (1% acetic acid) at the time points (30, 60, 120, and 180 s).
Extracts were separated by centrifugation, dried, and analyzed
by HPLC. The amount of product in each aliquot was deter-
mined by comparing the integrated HPLC peak area to a stan-
dard curve prepared with known product concentrations. The
resulting initial velocity data were fit to the Michaelis-Menten
equation to compute the resulting kinetic parameters, kcat and
All models were validated with the following structure vali-
dation tools: PROCHECK (48), ERRAT (49), and VERIFY3D
(50). PROCHECK reports that 90.6% of the residues are in the
most favored region of the Ramachandran plot; 9.1% of the
residues are in additionally allowed regions; Ala-207 from chain
B is in a generously allowed region, and Asp-173 is in a disal-
lowed region. Asp-173 appears in clear electron density. Its
unfavorable Ramachandran angles appear to be stabilized by
eight hydrogen bonds with neighboring residues. It immedi-
ately precedes the active site Ser-174 and might be involved in
catalysis. ERRAT reported that 96.9% of the residues were
within the 95% certainty limit for rejection. The coordinates of
the final model and the merged structure factors have been
deposited with the Protein Data Bank. The corresponding PDB
code is 3SKV.
KM
.
Chemoenzymatic Preparation of SF2575 Analogs—Analogs
of compound 6 were prepared by enzymatic reaction. Six 1-ml
reactions were prepared as follows: 50 mM HEPES, pH 7.9, 10
mM MgCl2, 0.5 mM compound 4, 2 mM free coenzyme A, 2 mM
ATP, 2 mM acid substrate, 15 ␮M SsfL1, and 5 ␮M SsfX3. Reac-
tions were incubated overnight and extracted with ethyl acetate
containing 1% acetic acid. Organic extract was dried, and the
final product was purified from the redissolved extract by
Size Exclusion Chromatography—Size exclusion chromatog-
raphy analysis of SsfX3 was performed on a Superdex 75 (10/ HPLC.
41542 JOURNAL OF BIOLOGICAL CHEMISTRY
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Tetracycline Acyltransferase SsfX3
TABLE 1
tion and translation function. Phases were further improved by
2-fold symmetry averaging, and the final model was refined
against native diffraction data to 2.5 Å resolution. The model is
mostly complete; of the 384 residues that comprise SsfX3, it was
possible to model residues 8–93 and 97–364 in chain A, and
residues 12–85, 97–272, and 283–364 in chain B. Refinement
statistics are reported in Table 1.
Statistics of data collection, phasing, and refinement
The highest resolution shell is shown in parenthesis.
Overall Architecture of SsfX3—From the structure, shown in
Figs. 2A and 4A, it is clear that SsfX3 is indeed composed of two
domains. A flexible linker divides the N-terminal ␤-sandwich
domain and the C-terminal GDSL hydrolase region as pre-
dicted by sequence comparison to the consensus GDSL hydro-
lase sequence. It also contains a large open binding pocket,
which is bounded by the aromatic residues Phe-276, Trp-277,
and Tyr-335 from the C-terminal domain at one end and the
N-terminal ␤-sandwich domain on the other end (Fig. 2 and 4).
The N-terminal region consists of two short ␤-strands ␤1 and
␤2 followed by a 10-stranded ␤-sandwich or “jelly roll” fold.
Between ␤4 and ␤5 in the ␤-sandwich domain is a loop contain-
ing two antiparallel 310 helices A and B. The C-terminal domain
displays the atypical ␣/␤-hydrolase fold characteristic of GDSL
hydrolases (12). The ␤-strands ␤13–17 are arranged in a paral-
lel ␤-sheet with a slight twist. This ␤-sheet formation is flanked
by five ␣-helices, two on the concave surface (␣C and ␣K) and
three on the convex surface (␣E, ␣G, and ␣I). There is also a
short helix ␣F in the loop connecting ␤15 and ␣G, which bor-
ders the active site. Another short helix ␣J directly follows ␤17
and precedes the 12-residue loop ending at the general base
His-338 in ␣K.
Bioactivity Assays—Cell proliferation was determined by
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium assay for adherent HeLa and
M249 cells and suspension of Nalm-6 and Jurkat cells. All cells
were cultured according to ATCC or DSMZ standards. Jurkat
and Nalm-6 cells were seeded at 8 ϫ 104 cells/well, and HeLa
cells were seeded at 4 ϫ 103 cells/well. Compounds of interest
dissolved in DMSO were added to wells after 12 h to a final
concentration ranging from 10 nM to 10 ␮M. Cell proliferation
was assayed 72 h after treatment using CellTiter 96ꢁ AQueous
OneSolution cell proliferation assay reagent (Promega) and
measured with a PowerWave XS 96-well plate reader at 490 nm.
Values were compared with control cells plus 0.1% DMSO.
A structural similarity search using the Dali server (51)
revealed five structures having three-dimensional similarity
over both the ␤-sandwich and the ␣/␤-hydrolase-fold domains
of SsfX3. All of these were from the CE2 family of carbohydrate
esterases, which includes CtCE2 (38). Despite their low
sequence identity (less than 16% for CtCE2), they were well
aligned across both domains with a root mean square deviation
(r.m.s.d.) of 2.9 Å and 282/327 residues aligned. These struc-
tures were all reported by Montanier et al. (38) as acetyles-
terases acting on noncellulosic plant polysaccharides. Addi-
tional enzymes were identified that independently mapped to
either the ␤-sandwich or the ␣/␤-hydrolase domains. The
RESULTS AND DISCUSSION
Structure Determination and Model Quality—The crystal C-terminal region, as expected, shares structural similarity with
structure of a selenomethionine-containing SsfX3 mutant other GDSL hydrolases such as TAP (13) with r.m.s.d. of 2.4 Å
C68H was solved using the MAD method. The C68H mutant with 172/178 residues structurally aligned. Proteins with struc-
was prepared to eliminate nonspecific oxidation of the free tural similarity to the N-terminal jelly roll domain of SsfX3
thiol. Size exclusion chromatography was used to determine include members from the CBM6 (52) and CBM35 (53) families
that SsfX3 was a monomer in solution. Because of severe radi- of type B CBMs (54). CBM35 (Pel-CBM35, PDB code 2W3J)
ation sensitivity of the crystals, two isomorphous crystals were (53) is structurally similar to the N-terminal domain of SsfX3
used for x-ray data collection, one at the peak wavelength and with a 2.5-Å r.m.s.d. and 115/137 residues aligned. CBM mod-
the other at the inflection and remote wavelengths (see under ules are often found appended to catalytic domains to promote
“Experimental Procedures”). Statistics for experimental phases substrate interaction; an example is Pel-CBM35, which
calculated to 2.9 Å are reported in Table 1. The map was of together with a separate catalytic pectin lyase domain com-
sufficient quality to allow manual placement of several poses the multidomain enzyme Pel10 (53).
␤-strands and ␣-helices, from which it was possible to recog-
There is a large loop region between ␤4 and ␤5 of the N-ter-
nize structural similarity with the family 2 carbohydrate ester- minal domain (residues 41–61) that contains two short 310 hel-
ase from Clostridium thermocellum (PDB code 2WAB) (38). ices ␣A and ␣B. This insertion in the ␤-sandwich motif is not
With newly acquired phases, two molecules of the esterase found in either the Pel-CBM35 or the CtCE2 structures, as
could be placed in the asymmetric unit using the phased rota- shown in Fig. 2C. Interestingly, this region appears to be con-
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Tetracycline Acyltransferase SsfX3
41544 JOURNAL OF BIOLOGICAL CHEMISTRY
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Tetracycline Acyltransferase SsfX3
TABLE 2
Catalytic activity of SsfX3 mutants
served among proteins with higher sequence similarity, as
shown in Fig. 2D, including AviX9 and Cpz23 from other nat-
ural product pathways, yet is missing from CtCE2. Instead, in
the same spatial region (residues 679–693) of CtCE2 are two
short ␤-strands inserted between ␣C and ␤15 of the ␣/␤-hy-
drolase domain (Fig. 2, B–D). Additional loop regions that are
structurally distinct between SsfX3 and CtCE2 are shown in
Fig. 2, C and D. CtCE2 contains a large insertion following the
GDSI active site motif, which results in a much larger loop than
that found in SsfX3. This larger loop effectively blocks off part
of the active site, resulting in a much narrower binding pocket
in CtCE2 compared with SsfX3, and it also reduces the contact
area of the N-terminal domain with the binding pocket. This
larger loop in CtCE2 and the aforementioned ␤-strand inser-
tion (residues 679–693) results in a binding pocket that is
defined almost entirely by the C-terminal ␣/␤-hydrolase
domain. In contrast, the SsfX3 binding pocket is outlined by
numerous residues from both N- and C-terminal domains, in
particular ␣A and ␣B, which extend into the active site, thereby
increasing the contribution of the N-terminal domain to the
active site surface area (Figs. 2 and 4).
K
M of
K of
Mutant
salicylyl-CoA 5
tetracMycline 4
kcat
min
46 Ϯ 5.8
Ϫ1
␮M
␮M
SsfX3 WT
S174A
H338A
D333A
E330A
C176A
C176S
I175N
F276A
W277A
Y335A
L142N
Q61A
41 Ϯ 11
Յ5a
b
b
b
b
b
b
27 Ϯ 2.5
Յ5a
24 Ϯ 0.7
Յ0.1
c
c
c
c
b
c
c
b
Յ0.1
Յ0.1
b
12 Ϯ 4.1
35 Ϯ 1.3
12 Ϯ 4.6
42 Ϯ 2.6
215 Ϯ 55
941 Ϯ 363
91 Ϯ 8.0
15 Ϯ 3.4
40 Ϯ 8.6
32 Ϯ 9.1
18 Ϯ 6.0
11 Ϯ 0.8
24 Ϯ 7.6
Յ5a
11 Ϯ 1.2
9.7 Ϯ 0.1
30 Ϯ 3.1
5.3 Ϯ 0.1
3.4 Ϯ 0.4
2.6 Ϯ 0.4
46 Ϯ 2.1
32 Ϯ 1.0
19 Ϯ 1.3
R58L
Յ5a
P85W
Q93L
Յ5a
Յ5a
E97A
Յ5a
a Lower concentrations were below the assay detection limit.
b No activity was observed.
c Conversion was too low to obtain accurate KM values.
The D333A mutant, however, retained catalytic activity with a
Identification of Catalytic Residues—The active site of kcat of approximately half of the wild type enzyme (Table 2).
canonical ␣/␤-hydrolases includes a highly conserved catalytic Once the structure was solved, it became clear that Asp-333
triad consisting of a nucleophile, an acid, and a general base was not positioned with respect to Ser-174 and His-338 in an
histidine (11). For the GDSL hydrolase subfamily, this typically orientation compatible with catalysis (see Fig. 2A). Asp-333
manifests as a Ser-Asp-His catalytic triad (12), which was pre- resides on the far end of the loop between ␤17 and ␣K with its
dicted to be Ser-174, Asp-333, and His-338 in SsfX3 based on side chain extending away from, rather than pointing into, the
sequence homology. Additionally, the GDSL hydrolase family active site. The carboxylate chain of Glu-330, however, extends
lacks the GXSXG motif that results in a tight strand-turn-helix toward the active site in an orientation similar to that observed
motif characteristic of the geometry of the “nucleophile elbow.” for other known GDSL hydrolase structures as shown in Fig.
␦1
Instead, the active serine is characteristically embedded in a 3A. The 2.8-Å distance between Glu-330 O⑀2 and His-338 N
Gly-Asp-Ser-Leu (GDSL) motif lacking the tight turn confor- made it a more likely candidate as the third member of the triad.
mation. SsfX3 contains a slightly altered active site motif, con- Indeed, the mutant E330A displayed near complete attenuation
sisting of Gly-Asp-Ser-Ile (GDSI), which resides in a 15-residue in catalytic activity (less than 0.1% of wild type). Based on these
loop region (residues 173–187) between ␤13 and ␣C of the results, the active site of SsfX3 is verified to consist of the
C-terminal domain as seen in Fig. 2A. To verify the catalytic nucleophilic Ser-174, general base His-338, and the carboxylate
triad of SsfX3, kinetic assays were performed using a discontin- Glu-330 (Fig. 3A).
uous time course assay. The salicylyl transfer reaction contain-
Interestingly, these catalytic residues lie significantly further
ing substrates 4 and salicylyl-CoA 5 was initiated by the addi- apart from each other in SsfX3 than they do in homologous
tion of SsfX3, and aliquots at different time points were esterases (Fig. 3A). With no substrate bound, the distance
extracted and analyzed by HPLC. The amount of product 6 between Ser-174 O and His-338 N⑀2 is 8.6 Å. The enzyme must
␥
formation was quantified through integration of the HPLC therefore undergo a significant conformational change upon
peak area, and concentrations were calculated based on a stan- substrate binding to optimally position the general base within
dard line fit. Wild type SsfX3 was found to have a kcat of 46 Ϯ 5.8 hydrogen bonding distance to the nucleophilic hydroxyl (55).
minϪ1 and a KM for compound 5 of 41 Ϯ 11 ␮M. The KM value Compared with other GDSL hydrolases, this distance between
of SsfX3 toward compound 4 could not be reliably measured the Ser-174 and His-338 is larger than in the E. coli TAP (19, 20)
below 5 ␮M due the limitations of HPLC detection. Each of the and the three known CE2 esterases (38); those structures show
predicted catalytic triad residues was mutated to alanine to distances in the range of 2.7–3.8 Å for the unbound structures.
␦1
confirm its role in catalysis. Neither of the S174A and H338A As expected for catalytic triads in esterases, the His-338 N
mutants had detectable activity for either the forward salicylyl proton is within hydrogen bonding distance (2.7 Å) to the car-
⑀2
transfer reaction or the reverse hydrolysis of compounds 6 to 4. boxylate of Glu-330 O . An unusual feature of the SsfX3 active
FIGURE 2. A, overall structure of SsfX3 highlighting residues discussed in the text. B, structure of CtCE2 complexed with cellohexaose (PDB code 2WAO) (38).
C, structural alignment of SsfX3 and CtCE2. Where the structures diverge, green ribbons indicate the backbone trace of CtCE2 and purple ribbons indicate the
backbone trace of SsfX3. D, sequence alignment of SsfX3 with CtCE2 and other homologs identified based on sequence similarity. Homologs shown are AviX9
from Streptomyces viridochromogenes (accession number AAK83171), a putative GDSL family lipolytic protein from Streptomyces bingchenggensis (accession
number ADI04849), a putative GDSL family lipolytic protein from Paenibacillus sp. Y412MC10 (accession number YP_003243452), LipT from Streptomyces sp.
SANK 60405 (accession number BAJ05896), Cpz23 from Streptomyces sp. MK730–62F2 (accession number ACQ63631), and CtCE2 from C. thermocellum
(accession number 2WAB_A).
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Tetracycline Acyltransferase SsfX3
FIGURE 3. A, active site of SsfX3 showing side chains of catalytic residues Ser-174, Glu-330, and His-338, residues proposed to be involved in stabilizing the
anionic tetrahedral transition state (Ala-207 and Ser-235), and Cys-176, which was shown to be required for catalysis. B, active site of uncomplexed E. coli TAP
(PDB code 1JRL) (13) showing the catalytic triad Ser-10, Asp-154, and His-157 and oxyanion hole residues Gly-44 and Asn-73 (13). C, active site of uncomplexed
Cellvibrio japonicas CjCE2A (PDB code 2WAA) (38) showing catalytic triad Ser-160, Asp-333, and His-335 and oxyanion hole residues Gly-205 and Asn-255.
D, proposed mechanism of acyl transfer catalyzed by SsfX3 and the roles of oxyanion hole residues.
site, however, is the number of residues separating the aspartate strate is bound. The two molecules were much closer in con-
and histidine. In nearly all GDSL hydrolases, the carboxylate formation over the remainder of their structures, with an over-
residue (usually aspartate) and the histidine are separated by all r.m.s.d. of 1.2 Å.
only one or two residues (12). In the case of SsfX3, residues
Residues Ile-175 and Cys-176, which immediately follow the
Glu-330 and His-338 are separated by a seven-residue flexible catalytic serine (GDSI) and line the bottom of the binding
loop. Glu-330 resides at the start of the loop following a short pocket, were mutated to determine their effects on kinetic
3
10 helix ␣H, and His-338 is positioned at the end of the loop parameters (Fig. 2A). Because it is unusual for GDSL hydrolases
immediately before ␣K. This large loop may allow both Glu-330 to contain an isoleucine following the nucleophilic serine, an
and His-338 more flexibility to undergo large scale conforma- I175N mutant was prepared to determine whether a nonpolar
tional changes required to form the required hydrogen bond residue is a requirement at this position. The resulting mutant
network with Ser-174 upon substrate binding. The flexibility of had no observable acyltransferase activity, suggesting that a
this active site loop is supported by the structural variations hydrophobic residue is indeed required there for enzyme activ-
observed in this region between the two SsfX3 molecules in the ity. In the early years following the discovery of this class of
␥
asymmetric unit. In the other SsfX3 molecule, the Ser-174 O
␣/␤-hydrolases, the conserved active site motif was defined as
⑀2
⑀2
and His-338 N distance is 12.9 Å, whereas the Glu-330 O
GDSLS, because a serine is frequently found in this fifth posi-
and His-338 N␦1 distance is 4.17 Å. The variation between these tion. A S31A mutation in EstA arylesterase from Vibrio mim-
two independent molecules is mostly localized to the active site, icus decreased the KM by a factor of 2 compared with the wild
which further points to flexibility in this region when no sub- type enzyme and had little effect on the catalytic rate (56). In
41546 JOURNAL OF BIOLOGICAL CHEMISTRY
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Tetracycline Acyltransferase SsfX3
contrast, mutation of S18G or S18V in GCAT from Aeromo- (Fig. 4, B and C). During biochemical analysis of CtCE2, a pre-
nas hydrophila resulted in complete inactivation of the vious investigation found that aromatic residues lining the top
enzyme (57). Unlike the homologous GDSL enzymes shown and bottom of the binding pocket were important for substrate
in Fig. 2D, SsfX3 has a cysteine residue in this position. The interactions (38). In the active site pocket of SsfX3, three aro-
Ϫ1
mutant C176A resulted in reduction of kcat to Յ0.1 min
.
matic residues (Phe-276, Trp-277, and Tyr-335) form one end
Interestingly, despite the common occurrence of a serine of the binding pocket as seen in Figs. 2A and 4B. The aromatic
following the GDSL motif, the C176S mutant also showed side chains of Tyr-335 and Trp-277 line the top and bottom of
greatly reduced activity (Table 2). Therefore, the cysteine the cavity, similar to Trp-790 and Trp-746 in CtCE2, respec-
thiol is highly important for SsfX3 catalysis; its specific tively (Fig. 2). Phe-276 forms a wall at the end of the binding
role is presently unknown, but it may be involved in sub- pocket, thereby making it an enclosed cavity. The helix ␣H
strate interaction or in somehow maintaining the correct where Phe-276 is found in SsfX3 is missing in CtCE2. As a
active site configuration. As a note, CtCE2 also contains a result, the binding pocket of CtCE2 is more like an open-ended
cysteine of unknown function, Cys-616, in close proximity to channel (compare Fig. 2, A and B). These aromatic residues in
the active serine (Fig. 2D).
SsfX3 were mutated to alanine. The resulting kinetic parame-
In addition to the catalytic triad, GDSL hydrolases are iden- ters are shown in Table 2. F276A and W277A both resulted in
tified by four invariant residues (Ser, Gly, Asn, and His) making an ϳ4-fold decrease in kcat. As neither of those residues is pre-
up the oxyanion hole. These residues lie in conserved blocks dicted to be involved in catalysis, the diminished activity is
I–III and V and lead to the alternative designation of “SGNH likely due to effects on the conformation of nearby residues
hydrolases” for this family of enzymes (12). Because of the low caused by removing a bulky side chain from the active site. All
sequence similarity of SsfX3 to other GDSL hydrolases, only three mutants F276A, W277A, and Y335A resulted in an
blocks I and III are conserved in SsfX3 and only weakly. Based increase in the KM value for compound 4. On the contrary,
on sequence alignment (Fig. 2D), residues that make up the Y335A and F276A resulted in an ϳ4-fold lower KM value
oxyanion hole were initially predicted to be Ser-174, Gly-209, toward the acyl donor 5. KM values for compound 5 remained
Asn-236, and His-338 (9). A pairwise structure comparison relatively unchanged for W277A. These results indicate that
using the DaliLite server with TAP and CtCE2 revealed that the aromatic residues F276A, W277A and Y335A play impor-
Gly-209 is distant from the active site and that the backbone tant roles in binding the polycyclic tetracycline substrate 4, but
amide of Ala-207 is more likely to contribute to the oxyanion may not be involved in the binding of compound 5.
hole. The spatial arrangement of Ala-207 is also nearly identical
Role of the N-terminal ␤-Sandwich Domain—As GDSL
to that of Gly-44 of TAP (19) and Gly-658 of CtCE2 (38). The hydrolases are often isolated as single domain enzymes without
peptide backbone of Asn-236 does align closely with the afore- fusion to the ␤-sandwich domain, we were interested to deter-
mentioned structures. However, the side chain extends away mine whether the C-terminal hydrolase domain had any activ-
rather than into the active site, and rotation of the side chain ity as a stand-alone protein or whether it required the addi-
amide to accommodate the oxyanion appears unlikely. The side tional ␤-sandwich domain for which no catalytic activity was
chain of the neighboring residue Ser-235 is facing the active site predicted. The excised C-terminal hydrolase domain, starting
and may be a more likely candidate to serve as a hydrogen bond from residue Thr-162 in the linker region, was expressed in
donor to the oxyanion. A sequence alignment with homologs of E. coli at yields comparable with wild type SsfX3 (ϳ60 mg/liter).
SsfX3 shows that Ser-235 is aligned with asparagine residues in Neither the forward transcylation nor the reverse hydrolysis
four of the six homologs examined (Fig. 2D). Additionally, activities were detected with the stand-alone enzyme, demon-
homologs Cpz23 and LipT contain nonpolar residues at this strating that the N-terminal domain is vital to the activity of
position, suggesting that the oxyanion hole residues may not be SsfX3. The additional domain may be needed to provide suffi-
strictly conserved.
cient binding energy for large substrates such as compound 4.
Site-directed Mutagenesis of Binding Pocket Residues—Un- The canonical, single domain acyltransferases may not have
fortunately, we were unable to obtain a co-crystal structure sufficient binding interactions to confer substrates specificity
with either compounds 4 or 6 to identify the exact residues and for the large substrate.
dimensions of the binding pocket. Attempts to model substrate
To further examine the role of the N-terminal domain, resi-
binding using computational docking software were also dues that reside at the interface of the two domains, and in close
unsuccessful, likely due to the large conformational changes proximity to the putative binding pocket (Fig. 4), were mutated
predicted to occur upon substrate binding. To map regions that to determine whether they had an impact on reaction kinetics
are potentially important to substrate binding, we instead uti- or substrate affinity. During analysis of CBM35, Montanier et
lized site-directed mutagenesis to experimentally probe the al. (53) found that binding of carbohydrates to CBM35 is facil-
role of individual regions in and around the putative binding itated by a combination of hydrophobic interactions involving
pocket. All of the mutants were solubly expressed and purified several aromatic residues and hydrogen bonding from polar
from E. coli at levels similar to that of the wild type SsfX3 (ϳ60 side chains. In the ␤-sandwich domain of SsfX3, however, no
mg/liter culture).
aromatic residues were positioned in close proximity to the
Because of the large size of the tetracycline substrate 4, which likely substrate binding pocket. Instead, the side chain of Leu-
spans ϳ16 Å from end to end (ϳ20 Å for the product 6), the 142 positioned at the beginning of ␤11 inserts into the inter-
substrate is expected to extend from the active site pocket of the domain binding pocket and may be oriented for contact with
␣/␤-hydrolase domain to the interface of the ␤-sheet domain the hydrophobic portions of the tetracycline substrate 4, such
DECEMBER 2, 2011•VOLUME 286•NUMBER 48
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Tetracycline Acyltransferase SsfX3
FIGURE 4. A, schematic view of SsfX3 highlighting its bidomain architecture and linker region. Residues discussed in the text are shown as sticks in left panel,
which highlights the active site. Right panel is rotated 90° to show the back side of the molecule and the linker between the N- and C-terminal domains.
B, space-filling representation of SsfX3 showing the size of the binding pocket and location of residues affecting substrate affinity. The active site Ser-174 is
shown in red; hydrophobic residues Leu-142, Phe-276, Trp-277, and Tyr-335, which are important for tetracycline substrate 4 affinity, are shown in orange, and
residues Pro-85, Gln-61, and Arg-58, mutation of which caused increases in KM for salicylyl-CoA 5, are shown in blue. C, SsfX3 surface representation with the
N-terminal CBM domain colored in red, the C-terminal ␣/␤-hydrolase domain colored in blue, and the active site Ser-174 shown in yellow. The white arrow
represents an approximation of the binding pocket, which contains residues from both N- and C-terminal domains.
as the aromatic D-ring or D-olivose. As expected, mutating Leu- protrude into the putative binding pocket and may form hydro-
142 to the polar residue asparagine resulted in a significantly gen bonds with either substrate 4 or 5. These residues were
increased KM for compound 4 and a 10-fold reduction in kcat
.
mutated to nonpolar amino acids, and the kinetic parameters
The L142N mutation did not affect the KM value for compound were evaluated (Table 2). Neither E97A nor Q93L mutants,
5, suggesting that this residue is not involved in binding the acyl which reside on the large flexible loop between ␤6 and ␤7,
donor. The side chains of Arg-58, Gln-61, Gln-93, and Glu-97 showed a significantly negative effect on enzyme activity. The
41548 JOURNAL OF BIOLOGICAL CHEMISTRY
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Tetracycline Acyltransferase SsfX3
TABLE 3
Catalytic activity of SsfX3 toward varies acyl donors
E97A mutant had no measurable effect on the KM value of
either substrate but resulted in an ϳ2-fold decrease in kcat. The
Q93L mutant had a slightly decreased kcat and an ϳ3-fold
reduction in KM for compound 5.
Substrate
KM
␮M
kcat
min
Relative kcat/KM
Ϫ1
Salicylyl-CoA compound 5 41 Ϯ 11
46 Ϯ 5.8
2.7 Ϯ 0.2
2.0 Ϯ 0.08
1.0
Arg-58 and Gln-61 both lie in the large loop region between
␤4 and ␤5, which contains the two 310 helices that line the
bottom of the putative binding pocket (Fig. 4B). Mutation of
these residues had the largest effect on kinetic parameters. The
Q61A mutant showed a reduction in kcat of ϳ10-fold and a
Benzoyl-CoA
75 Ϯ 18
11 Ϯ 1.4
0.032
0.16
2-Chlorobenzoyl-CoA
2,3-Dihydroxybenzoyl-CoA 357 Ϯ 39
1.4 Ϯ 0.005 0.0035
Butyryl-CoA
922 Ϯ 194 0.54 Ϯ 0.04 0.0005
decrease in affinity for compound 5 with a 4-fold increase in KM, CoA. Additional substitutions around the ring led to unfavor-
although the KM value for compound 4 remained below detec- able binding to SsfX3, as noted by the much higher KM value for
tion limits. The R58L mutant had a similar reduction in kcat and 2,3-dihydroxybenzoyl-CoA. This indicated that the SsfX3
an even more pronounced increase in KM for compound 5 of binding pocket affords a tight fit to the aryl functionality. Sur-
greater than 20-fold. Pro-85 also lies in this region at the inter- prisingly, however, SsfX3 displayed measurable activity toward
face of the N-terminal domain and putative substrate binding the butyryl group, as the C-4-butyryl analog of 6 was detected in
pocket. The proline residue was mutated to a tryptophan to the transacylation assay using butyryl-CoA. Despite the signif-
determine whether reducing the size of the binding pocket icantly reduced reaction velocity, this result demonstrated that
might influence the kinetic parameters. The kcat and KM values the aromatic moiety is not an absolute requirement for SsfX3.
for compound 4 remained unchanged, whereas the KM value Unfortunately, in the absence of a cocrystal structure, the spa-
for compound 5 was twice the wild type value. These mutagen- tial arrangement of the binding pocket cannot be mapped in
esis results hint at a possible difference in binding locations for detail.
compounds 4 and 5. Pro-85 lies adjacent in space to Gln-61 and
Arg-58 and may further define the binding region for the acyl ferases with respect to the acyl acceptor are assayed less fre-
donor. quently, which may be due in part to difficulties in preparing or
The substrate specificities of natural product acyltrans-
These results indicate that the N-terminal domain has an isolating analogs of the natural substrate to test. In the case of
important role in substrate binding for both the tetracycline ansamitocin biosynthesis, Asm19 showed broad substrate
substrate 4 and acyl donor 5. In particular, we noticed that the specificity toward various acyl substrates but was unable to
surface area of the N-terminal domain in the putative binding acylate the C-3 hydroxyl of related substrate maytansinol (58).
pocket was enlarged by the aforementioned 310 helices ␣A and N-Acyltransferases tAtf and aAtf, which acylate the sugar
␣B. Although this structural feature was not observed for car- amino groups during the biosynthesis of glycopeptides teico-
bohydrate esterase CtCE2, the region containing the two 310 planin and A-40,926 respectively, demonstrated flexibility for
helices appears to be present in AviX9 and Cpz23. Interestingly, both the acyl substrates and acyl acceptors (3). To examine the
all of these bidomain GDSL hydrolases are predicted or known substrate specificity of SsfX3 toward the aryl acceptor, we
to bind large extended substrates (although all very different assayed the transacylation reaction using upstream intermedi-
from compound 4). Hence, recruitment of the additional ates 1 and 2 (Fig. 1) in the presence of 5. Compound 2 differs
␤-sandwich domain by the GDSL catalytic domain may be a from 4 in that the C-ring has not been oxidized and is an analog
general strategy used to define the binding pocket for the of anhydrotetracycline instead of tetracycline. Compound 2
“large” small molecule substrates such as compound 4, to pro- also lacks the C-6 O-methoxy substitution present in com-
vide sufficient interactions and binding energies required for pound 4 and is therefore nearly isosteric to compound 4 but
forming the enzyme-substrate complexes.
with important physical differences. Compound 1 is the pre-
Substrate Specificity of the Acyl Group and Hydroxyl Donor— cursor of compound 2 and lacks the C-9 D-olivose group. From
Broad substrate specificity with regard to the acyl donor is com- active site mutations such as L142N, we reasoned that the distal
monly observed for acyltransferases (12), and this flexibility has end of compound 4, which includes the C-glycosylated D-oli-
been exploited in several cases to generate analogs of natural vose, makes van der Waals contacts with the N-terminal CBM-
products with varied acyl substituents (2, 3, 5, 58–60). SsfX3 like domain. Therefore, we anticipated that the smaller com-
has been shown previously to accept a variety of acyl-CoA sub- pound 1 would be a poor substrate for SsfX3. Product assays
strates, including those containing small substitutions around were performed with 0.1 mM compounds 1, 2, or 4, 0.3 mM
the aromatic ring, such as 2-chlorobenzoyl-CoA, 2,3-dihy- compound 5 and 1 ␮M SsfX3. Reactions were incubated at
droxybenzoyl-CoA, 2,4-dihydroxybenzoyl-CoA, and 2,5-dihy- room temperature for 1 h, extracted, and analyzed by LCMS.
droxybenzoyl-CoA. Larger groups such as 4-aminobenzoyl- Both compounds 1 and 2 were salicylated by SsfX3, albeit at a
CoA were shown not to be tolerated, resulting in no detectable much slower rate than the native substrate 4. As expected, com-
analog of compound 6 (9). The kinetic parameters of SsfX3 with pound 1 had the lowest conversion as follows: only 1.6% in 1 h
respect to several of the different aryl-CoA substrates were compared with 17% for compound 2 and 90% for native sub-
determined (Table 3). Although the kcat value for each of the strate 4 (supplemental Fig. S1). Although not tested in vitro,
analogs was at least 20-fold lower than that observed for trace amounts of SF2575 variants lacking either the C12a or C-6
compound 5, the KM values varied. Substitution at the ortho O-methylation have been identified in the fermentation extract
position is clearly preferred, as the KM value for both 2-chloro- of strains producing SF2575, indicating that these demethyl
benzoic acid and compound 5 were lower than that of benzoyl- analogs of compound 4 are also accepted as substrates of SsfX3.
DECEMBER 2, 2011•VOLUME 286•NUMBER 48
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Tetracycline Acyltransferase SsfX3
TABLE 4
tion of analogs of SF2575 intermediate 6 for structure-activity
relationship studies. Substrate specificity was more stringent
for the acyl acceptor, as alternative tetracycline substrates
resulted in significantly lower conversion to acylated products.
This increased substrate specificity compared with other mem-
bers of the GDSL hydrolase family, along with structural lines of
evidence, strongly indicates a role for the N-terminal ␤-sand-
wich domain in substrate recognition. This study therefore
sheds light on the function of SsfX3 and sets the stage for devel-
opment of this enzyme as a biocatalyst for diversifying tetracy-
Bioactivity data for C-4 substituted analogs of compound 3
C-4 aryl substituent
HeLa IC50
Nalm-6 IC50
Jurkat IC50
nM
nM
nM
Salicylate compound 5
Benzoate
510
220
110
700
240
160
2-Chlorobenzoate
520
275
110
2,3-Dihydroxybenzoate
2,4-Dihydroxybenzoate
2,5-Dihydroxybenzoate
Ն10,000
Ն10,000
Ն10,000
870
500
6000
Ն10,000
5100
Ն10,000
These studies demonstrate that SsfX3 has somewhat relaxed cline scaffolds.
substrate specificity for both the acyl donors and acyl acceptors.
Acknowledgments—We thank Dr. Duilio Cascio, the staff of the
However, the substrate specificity observed for SsfX3, although
somewhat tolerant to closely related substrates, seems to be
much more stringent when compared with the extremely broad
range of substrates accepted by GDSL hydrolases reported by
Asler et al. (24). According to the crystal structure of SsfX3, this
increased substrate specificity is likely influenced significantly
by the putative N-terminal binding module, as discussed
previously.
Advanced Light Source beamline 8.2.1, and the staff of the Advanced
Photon Source, Northeastern Collaborative Access Team (NECAT)
beamline 24-ID-C for assistance with data collection. The NECAT
beamline is supported by National Institutes of Health Award
RR-15301 from NCRR. Use of the Advanced Photon Source is sup-
ported by the United States Department of Energy, Office of Basic
Energy Sciences under Contract W-31-109-ENG-38.
Bioactivity of Chemoenzymatically Prepared Analogs—As
SsfX3 had sufficient flexibility with regard to the acyl donor, we REFERENCES
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DECEMBER 2, 2011•VOLUME 286•NUMBER 48
JOURNAL OF BIOLOGICAL CHEMISTRY 41551

Enzymology:
Structural and Biochemical
Characterization of the
Salicylyl-acyltranferase SsfX3 from a
Tetracycline Biosynthetic Pathway
Lauren B. Pickens, Michael R. Sawaya, Huma
Rasool, Inna Pashkov, Todd O. Yeates and Yi
Tang
J. Biol. Chem. 2011, 286:41539-41551.
doi: 10.1074/jbc.M111.299859 originally published online September 29, 2011
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