Peptides from purified soybean b-conglycinin inhibit fatty
acid synthase by interaction with the thioesterase
catalytic domain
Cristina Martinez-Villaluenga
1
, Sanjeewa G. Rupasinghe
2
, Mary A. Schuler
2
and Elvira Gonzalez de
Mejia
1
1 Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, IL, USA
2 Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, IL, USA
Keywords
b-conglycinin-derived peptides; fatty acid
synthase; inhibitors; soybean; thioesterase
Correspondence
E. Gonzalez de Mejia, 1201 West Gregory
Drive, 228 ERML, MC-051, Urbana,
IL 61801, USA
Fax: +1 217 265 0925
Tel: +1 217 244 3196
E-mail:
(Received 19 August 2009, revised 7
January 2010, accepted 8 January
2010)
doi:10.1111/j.1742-4658.2010.07577.x
Fatty acid synthase (FAS) is uniquely expressed at high levels in cancer
cells and adipose tissue. The objectives of this study were to identify, purify
and validate soy FAS inhibitory peptides and to predict their binding

peptides from purified b-conglycinin hydrolysates and predict their binding
modes at the molecular level, leading to their possible use as nutraceuticals.
Structured digital abstract
l
MINT-7544766, MINT-7546418, MINT-7546830: Beta-conglycinin (uniprotkb:P25974) binds
(
MI:0407)toAlpha subunit of BC (uniprotkb:P13916)bypull down (MI:0096)
l
MINT-7547140, MINT-7547249: Beta-conglycinin (uniprotkb:P25974) binds (MI:0407)to
Beta subunit of BC (uniprotkb:
P25974)bypull down (MI:0096)
Abbreviations
ACP, acyl carrier protein; CIP, co-immunoprecipitation; DMEM, Dulbecco’s modified Eagle’s medium; ER, b-enoyl reductase; FAS, fatty acid
synthase; PDB, Protein Data Bank; SBC, soybean b-conglycinin; TBST, Tris-buffered saline containing 0.1% Tween 20; TE, thioesterase.
FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1481
Introduction
Fatty acid synthase (FAS, EC 3.2.1.85) is a multicom-
ponent enzyme that catalyzes the de novo biosynthesis
of long-chain fatty acids from acetyl-CoA and malo-
nyl-CoA through a NADPH-dependent cyclic reaction
[1]. FAS is homodimeric and each polypeptide chain
(270 kDa) carries seven catalytic domains integrating
all the steps needed for fatty acid synthesis [2,3]. The
growing fatty acid is covalently attached to an acyl
carrier protein (ACP), which transports it through the
active sites where each reaction is catalyzed. Once the
fatty acid reaches 16–18 carbon atoms in length, it is
released by the thioestherase (TE) domain [1].
Human FAS is downregulated in most normal
human tissues but is highly expressed in adipose and

b-conglycinin (SBC) contains active peptides that
inhibit FAS [23] and fatty acid biosynthesis in adipo-
cytes [24]. The objectives of this study were to iden-
tify SBC-derived peptides with FAS inhibitory
activity using co-immunoprecipitation (CIP) and pro-
teomic techniques. The FAS inhibitory activity of the
identified peptides was established in both biochemi-
cal assays and cell-based models of 3T3-L1 adipo-
cytes. The relationship between the chemical
characteristics of these peptides and their FAS inhibi-
tory potency was defined, and their binding modes
were predicted by docking simulations using the crys-
tal structures of mammalian FAS (PBD ID code:
2VZ8) [25] and human FAS (PBD ID code: 2PX6)
[17]. This study provides valuable information for the
rational design of new FAS inhibitors and the prepa-
ration of natural compounds potentially preventing
the development of cancer, obesity and related meta-
bolic disorders.
Results
Identification of FAS inhibitory peptides from
purified SBC hydrolysate
Previous work in our laboratory demonstrated that
hydrolysates from b-conglycinin with alcalase (Bacil-
lus licheniformis) exert a potent inhibitory effect
(IC
50
=30lm) on FAS activity [23]. In this study,
amino acid sequences, identified by LC-MS ⁄ MS, of
FAS inhibitory peptides co-immunoprecipitated from

C75. Interestingly, although the three synthetic pep-
tides showed FAS inhibitory activity, their potency
was different. RKQEEDEDEEQQRE exerted a strong
inhibitory activity ($ 40% inhibition) at doses as low
as 12 lm. To compare the potency of the FAS inhibi-
tory response across all of the peptides and the C75
positive control, classical sigmoidal dose–response
curves were plotted and used to calculate the IC
50
val-
ues listed in Table 2. In these, the larger peptides,
RKQEEDEDEEQQRE (IC
50
= 16.5 lm) and EI-
TPEKNPQLR (IC
50
= 27.4 lm), showed significantly
higher (P < 0.05) potency (lower IC
50
value) than the
C75 positive control (IC
50
= 80.3 lm). The smaller
KNPQLR peptide had a higher IC
50
value (79.9 lm)
(P < 0.05) more comparable with the positive control
C75 (P > 0.05).
Structure–potency relationship of FAS inhibitory
peptides

FAS activity inhibition (%)
KNPQLR
EITPEKNPQLR
RKQEEDEDEEQQRE
C75
Fig. 1. Fatty acid synthase (FAS) inhibitory activity of synthetic pep-
tides and C75. Synthetic peptides KNPQLR, EITPEKNPQLR and
RKQEEDEDEEQQRE inhibited FAS in a dose-dependent manner
which was similar to the positive control C75. Evaluation of FAS
activity was performed after 20-min preincubation with different
concentrations of each compound. Values were expressed as per-
cent inhibition of FAS activity compared with a negative control that
included no inhibitors. Each dataset corresponds to the mean of
three independent replicates with error bars indicating the standard
deviations. Different letters indicate significant differences at
P < 0.05 in one-way ANOVA analysis.
Table 2. Fatty acid synthase (FAS) inhibitory potency and physicochemical and biochemical characteristics of synthetic peptides. MM, pep-
tide molecular mass; pI, theoretical isoelectric point of each peptide; GRAVY, grand average of hydropathicity index. Parameters were
obtained using the Protparam tool in the ExPASY Proteomic Server.
Physicochemical properties
Biological activity
No. of charged
residues
Hydrophilic
amino
acids (%) GRAVY
Aliphatic
index
IC
50

C. Martinez-Villaluenga et al. b-conglycinin peptides inhibit fatty acid synthase
FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1483
higher potency), their molecular masses (r=+0.89)
and the number of negatively charged (r = +0.64)
and hydrophilic (r = +0.69) residues. By contrast, a
strong negative correlation was observed between their
potency and their pI values (r = )0.99). Together,
these results suggest that peptides with higher inhibi-
tory potency are larger and have more negatively
charged and hydrophilic residues. No correlations were
found between their IC
50
values and other physico-
chemical and biochemical parameters, such as the
number of positively charged residues, grand average
of hydropathicity index and aliphatic index.
Identification of the potential binding site of FAS
and inhibitory peptides from SBC
Peptides EITPEKNPQLR and RKQEEDEDEEQ-
QRE were selected for use in the ligand–enzyme
docking simulations because they displayed higher
FAS inhibitory potency. To identify potential binding
sites for these peptide inhibitors, the multidomain
porcine FAS crystal structure (PBD ID code: 2VZ8)
[25] was searched for cavities near the identified active
site residues in each domain. Because this structure
lacked the ACP and TE domains, the human ACP
structure (PBD ID code: 2CG5) and human TE
domain structure (PBD ID code: 1XKT) lacking three
short loop regions were also included in this search.

was performed using a recombinant human FAS TE.
The TE inhibitory activity displayed by peptides
EITPEKNPQLR and RKQEEDEDEEQQRE was
compared with C75 and Orlistat (Table 3). Soy pep-
tides were more potent (10 lm) than C75 (58.7 lm),
however, their potency was $ 10-fold lower
(P < 0.05) than Orlistat (0.9 lm). Similar to C75, soy
peptides blocked > 50% of the TE activity; however,
this inhibition was lower (P < 0.05) than Orlistat
(77.3%) at 100 lm.
Binding and interaction modes of FAS inhibitory
peptides
To evaluate in more detail the binding modes of
these peptide inhibitors in the TE domain, they were
KS
DH
KR
ER
ACP
TE
MAT
Fig. 2. Identification of active-site cavities in
fatty acid synthase (FAS). In this representa-
tion, the multidomain FAS is compiled in
MOE from the swine FAS crystal structure
(PDB ID code: 2VZ8) [25], the human ACP
structure (PBD ID code: 2CG5) [41] and the
human thioesterase (TE) domain structure
(PBD ID code: 1XKT) [1]. The protein back-
bone is represented as an orange line,

Asp 2338
His 2481
Ser 2308
Asp 2338
His 2481
Ser 2308
Asp 2338 His 2481
Ser 2308
Asp 2338
Subdomain A
Subdomain B
N
C
Loop II
Loop III
Loop I
A
C
B
D
Fig. 3. Predicted overall fold of the thioes-
therase (TE) domain with inhibitors bound.
The lowest-energy binding mode for
EITPEKNPQLR (A), RKQEEDEDEEQQRE
(B), C75 (C) and Orlistat (D) rendered in ball-
and-stick format in the human TE domain
model (backbone in tube format) is shown
with catalytic triad residues Ser2308,
His2481 and Asp 2338 in space-filling for-
mat. Details regarding docking simulations

b
55.46 ± 1.45
c
52.90 ± 4.34
c
55.53 ± 2.43
c
77.89 ± 0.81
d
Human FAS TE inhibitory potency (IC
50
, lM)
b
10.05 ± 1.60
c
10.71 ± 4.36
c
58.71 ± 6.74
e
0.93 ± 0.13
d
a
C75 and Orlistat were docked only in the active site of TE domain because they are known to target the TE domain [Cheng et al. [31]].
b
Compounds were tested at a concentration of 100 lM. Values indicate the mean ± SD of at least two independent experiments.
c,d,e
Dif-
ferent letters (c, d, e) in the same row indicate significant difference at P<0.05 in one-way ANOVA analysis.
C. Martinez-Villaluenga et al. b-conglycinin peptides inhibit fatty acid synthase
FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1485

)1
, respec-
tively) with the TE domain than was the smaller
SBC-derived peptide (Table 3). In addition, correla-
tion analyses between the inhibitory potency of pep-
tides EITPEKNPQLR, RKQEEDEDEEQQRE and
C75 and their interaction energies with the TE
domain showed a strong correlation (r = 0.99).
Close-up views of the binding modes of SBC-derived
peptides and Orlistat with the TE active site (Fig. 4)
suggest that the palmitic core of Orlistat is bound
almost exclusively to a hydrophobic groove generated
by subdomain B, and its peptidyl moiety is bound in
the interface cavity, whereas the hexanoil tail digs into
the short chain pocket where the catalytic triad exists.
These views also suggest that the larger EI-
TPEKNPQLR and RKQEEDEDEEQQRE peptides
bind throughout the long hydrophobic groove of the
TE domain in a orientation similar to that of Orlistat
with their amino acid side chains also extending into
the interface cavity and short chain pocket. The poten-
tial interaction modes of these peptides with the TE
domain suggest that EITPEKNPQLR (Fig. 5A) and
RKQEEDEDEEQQRE (Fig. 5B) bind mainly via
hydrophilic interactions (hydrogen-bonding and elec-
trostatic interactions) with active site residues. By con-
trast, only the hydrophilic peptidyl group of Orlistat
participates in hydrogen bonding with catalytic triad
residues Tyr2307, His2481 and Arg2482 located in the
interface cavity.

by these peptides correlated with FAS inhibition
(r = 0.70) (Fig. 6) even though the magnitudes of inhi-
bition in these peptides in the cell-based model were
lower than the magnitude of FAS inhibition measured
in biochemical assays; this is probably because of the
A
B
C
Fig. 5. Detailed 2D interactions between
inhibitors and the thioestherase (TE) domain.
Calculated using the
MOE program following
the method of Clark & Labute [44], residues
in the TE domain that contribute to the bind-
ing of EITPEKNPQLR (A), RKQEEDE-
DEEQQRE (B) and Orlistat (C) are shown
with green circles indicating residues with
no polar or charged side chains and light
mauve circles indicating polar side chains
that are either acidic (red ring) or basic (blue
ring). Arrows indicate hydrogen bonds to
side chain (green) and backbone (blue)
residues.
C. Martinez-Villaluenga et al. b-conglycinin peptides inhibit fatty acid synthase
FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1487
low permeability of cell to these longer peptides. No
effect on cell viability of 3T3-L1 adipocytes was
observed with any of the treatments used in this study,
indicating no cellular toxicity (data not shown).
Discussion

Molecular modeling has identified the TE domain as
the potential binding site for the FAS inhibitory pep-
tides from SBC. Molecular docking has shown that
soy peptides displayed a different inhibitory mecha-
nism than C75. Soy peptides are selective inhibitors of
the FAS TE domain, whereas C75 has been shown to
interact at several sites in FAS [14]. The predicted
binding energy of C75 in the FAS b-ketoacyl synthase
domain was )53.9 kcalÆmol
)1
, similar to that observed
in the TE domain ()51.2 kcalÆmol
)1
). These results
indicate that C75 is not a selective inhibitor for a
particular FAS domain, in agreement with previous
findings [14]. We also confirmed that the synthetic pep-
tides EITPEKNPQLR and RKQEEDEDEEQQRE
inhibited 4-methylumbelliferone heptanoate hydrolysis
by TE in in vitro experiments. Therefore, these
peptides are antagonists of TE under near physiologic
conditions, meaning that they bind to the unoccupied
enzyme and reduce substrate turnover. The TE domain
coordinates the terminal step of fatty acid synthesis by
hydrolyzing palmitate from the 4¢-phosphopanteine
arm of the ACP domain [1]. Its active site is comprised
of a hydrophobic groove with a distal pocket at the
interface of subdomains A and B and a hydrophilic
catalytic triad (Ser2308, His2481 and Asp2338) at the
proximal end of the groove [17]. Palmitate, the main

Inhibition (%) lipid accumulation
KNPQLR
EITPEKNPQLR
RKQEEDEDEEQQRE
C75
i
ghi
hi
fgh
ghi
fghi
fgh
def
fg
ef
cde
ab
bc
bcd
abc
a
Fig. 6. Inhibition of lipid accumulation in 3T3-L1 adipocytes by syn-
thetic peptides. 3T3-L1 adipocyte cells were treated with the syn-
thetic KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE peptides
at concentrations ranging from 0 to 100 l
M on days 3, 5 and 7, and
lipid accumulation was measured on day 10 using the Oil Red O
assay as outlined in Experimental procedures. Each dataset corre-
sponds to the average of three independent replicates with error
bars indicating the standard deviation. Different letters indicate sig-

through the inhibition of FAS appears to inhibit the
expression of neuropeptide Y which promotes inges-
tion [9]. Moreover, the FAS inhibitory activity of
C75 induced apoptosis and prevented the growth of
multiple tumor xenografts in vivo [36,37]. Our findings
clarify the mechanism linking FAS inhibition with the
anti-obesity effects of soy protein-derived peptides. In
conclusion, the soy peptides EITPEKNPQLR and
RKQEEDEDEEQQRE inhibited the TE domain and
de novo fatty acid synthesis in adipocytes. The bind-
ing mode of these peptides in the large palmitate-
binding pocket is of particular interest and will guide
future research. These FAS inhibitory peptides can
serve as lead compounds to design peptoid analogs
(oligomers of N-subtituted glycine) with equivalent
biological activity, enhanced systemic stability and
bioavailability than standard peptides [38]. The rele-
vance of the identification of these SBC-derived pep-
tides is noticeable because of the novelty of their
biological activity and chemical nature. Molecular
docking has allowed us to predict binding modes for
SBC-derived peptides (EITPEKNPQLR and RKQEE-
DEDEEQQRE) in the TE domain. Based on our
data, it is likely that the consumption of soy high in
b-conglycinin represents a preventive alternative to
improve health and wellness.
Experimental procedures
Materials
b-Conglycinin was purified from soybean defatted flour as
described in Wang et al. [39]. FAS inhibitory peptides were

NADPH and 0.3 lm FAS in 0.1 m potassium phosphate
buffer. Initial rates were calculated for the slope of the pro-
gress curves during the first 5 min.
FAS inhibition studies
Synthetic peptides and the C75 positive control compound
were used for FAS inhibition studies with stock solutions
of the synthetic peptides and C75 dissolved in deionized
water and dimetylsulfoxide, respectively, and serial dilutions
made in 0.1 m potassium phosphate buffer (pH 7.0). Inhibi-
tion studies were performed by measuring the residual FAS
activity after enzyme preincubation with inhibitors for
20 min at 37 °C. Potency was determined by dose–response
curves in which the range of concentrations was distributed
in a logarithmic scale and the IC
50
values were calculated
C. Martinez-Villaluenga et al. b-conglycinin peptides inhibit fatty acid synthase
FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1489
using nonlinear regression sigmoidal curve fit functions in
GraphPad prism 4.00 (Graphpad Software Inc., San Diego,
CA, USA).
Inhibition of FAS TE enzymatic activity was performed
using a fluorescence method described by Richardson &
Smith [41]. Peptides were added to yield a final concentra-
tion of 100 lm; in this assay the ability of the recombinant
TE to cleave 4-methylumbelliferone heptanoate and hydro-
lyzed it to the fluorescent 4-methylumbelliferone was
followed over time at 360 ⁄ 435 nm.
Co-immunoprecipitation
To purify FAS inhibitory peptides a CIP approach was

To confirm the CIP of FAS, western blot analysis was
carried out using goat polyclonal antibody (FAS IgG). Pro-
teins released from the beads were resuspended in Laemmli
loading buffer (BioRad, Hercules, CA, USA) containing
5% 2-mercaptoethanol. Samples (20 lg soluble protein)
were loaded onto 15% Tris ⁄ HCl ready gels and run
through a mini-electrophoresis kit at 200 V constant for
40 min. Further, proteins were transferred to poly(vinyli-
dene diflouride) membrane (BioRad) in blotting buffer
(25 mm Tris, 192 mm glycine pH 8.3, 0.1% SDS) using
western sandwich assembly for 1 h at 4 °C using 125 V.
After the transfer, membrane was blocked with 5% non-fat
dry milk in Tris-buffered saline containing 0.1% Tween 20
(TBST) for 1 h, followed by an overnight incubation with
goat polyclonal anti-(FAS IgG) (1 : 200) at 4 °C. Further,
membrane was washed with TBST four times and was incu-
bated with bovine anti-(goat IgG) horseradish peroxidase
conjugates (1 : 1000) for 1 h at room temperature. The
membrane was washed again in TBST for four times and
signals were visualized using chemiluminescence reagent
(GE Healthcare, Chalfont St Giles, UK) and a Kodak
Image Station 440 CF (Eastman Kodak Co., New Haven,
CT, USA).
LC-MS
⁄
MS
Samples were injected (10 lL) onto a dC
18
Atlantis nano-
Acquity column (75 · 150 mm, 3 lm particle size; Waters,

bovine serum, 0.5 mm isobutylmethylxanthine, 1 lm dexa-
methasone and 1.7 lm insulin (days 3 and 4). Cells were
then maintained in fetal bovine serum ⁄ DMEM with
1.7 lm insulin for another 2 days (days 5 and 6), fol-
lowed by culturing with fetal bovine serum ⁄ DMEM for
an additional 4 days (days 7–10), at which time > 90%
of cells were mature adipocytes with fat droplets. Cells
were treated on days 3, 5 and 7 of the differentiation
process with synthetic peptides dissolved in Dulbecco’s
phosphate buffer saline at a concentration ranging from
0to50lm and incubated at 37 °Cina5%CO
2
atmo-
sphere for 48 h.
b-conglycinin peptides inhibit fatty acid synthase C. Martinez-Villaluenga et al.
1490 FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS
Viability assay
The cell proliferation assay was conducted using CellTiter
96 Aqueous One Solution Proliferation assay kit (Promega
Corp., Madison, WI, USA) using 3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet-
razolium, inner salt, and an electron coupling reagent,
phenazine ethosulfate. Briefly, 5 · 10
3
preadipocytesÆwell
)1
were seeded in a 96-well plate and the total volume was
adjusted to 200 lL with growth medium. Cells were treated
on days 3, 5 and 7 of the differentiation process with differ-
ent concentrations of synthetic peptides dissolved in Dul-

control; 510 nm
À A
treatment; 510 nm

=
A
control; 510 nm  100 ¼ % inhibition of lipid content
Molecular modeling
To identify potential binding sites for the peptide inhibitors
the multi domain porcine fatty acid synthase crystal struc-
ture (PBD ID code: 2VZ8) [25] was searched for cavities
near the identified active site residues in each domain.
Because this structure lacked the ACP and TE domains,
the human ACP structure (PBD ID code: 2CG5) [42] was
used in the search. Parts of identified cavities extending to
the interdomain regions were excluded because they could
not be accounted for with the single domain structures. The
volumes of each of the identified cavities were calculated
using the SITE VOLUME SCRIPT function within moe.
The human TE domain structure available (PBD ID code:
1XKT) [1] lacked three loop regions: loop I (residues 2326–
2328 missing in chain A only) that connects a helix 4 (a4)
to b strand 5 (b5) and forms a surface loop on the under-
side of the a ⁄ b domain; loop II (residues 2344–2360) that
bridges subdomain A and subdomain B; and loop III (resi-
dues 2450–2460) that occurs near the catalytic triad linking
b6tob7. The missing loops were modeled using the
HOMOLOGY function in moe 2008.10 and the aligned
sequences of the TE domain of swine FAS (GenBank
accession no. NP_001093400) and human FAS (GenBank

(CSREES), AG 2007-34505-15767 Future Foods IL;
Illinois Soybean Association; the European Commis-
sion, Marie Curie IOF grant (PIOF-GA-2008-219860)
for Career Development (to CM-V). Special acknowl-
edgements to Drs J. W. Smith and R. D. Richardson
C. Martinez-Villaluenga et al. b-conglycinin peptides inhibit fatty acid synthase
FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1491
from the Burnham Institute for Medical Research,
California for providing us with the human recombi-
nant FAS TE.
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