Structure-based engineering of benzalacetone synthase.

Abstract

Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide synthases (PKSs), sharing 70% amino acid sequence identity and highly homologous overall protein structures. BAS catalyzes the decarboxylative coupling of 4-coumaroyl-CoA with malonyl-CoA to produce the diketide benzalacetone, whereas CHS produces the tetraketide chalcone by iterative condensations with three molecules of malonyl-CoA, and folding the resulting intermediate into a new aromatic ring system. Recent crystallographic analyses of Rheum palmatum BAS revealed that the characteristic substitution of Thr132 (numbering of Medicago sativa CHS2), a conserved CHS residue lining the active-site cavity, with Leu causes steric contraction of the BAS active-site to produce the diketide, instead of the tetraketide. To test this hypothesis, we constructed a set of R. palmatum BAS site-directed mutants (L132G, L132A, L132S, L132C, L132T, L132F, L132Y, L132W and L132P), and investigated the mechanistic consequences of the point mutations. As a result, the single amino acid substitution L132T restored the chalcone-forming activity in BAS, whereas the Ala, Ser, and Cys substitutions expanded the product chain length to produce 4-coumaroyltriacetic acid lactone (CTAL) after three condensations with malonyl-CoA, but without the formation of the aromatic ring system. Homology modeling suggested that this is probably caused by the restoration of the 'coumaroyl binding pocket' in the active-site cavity. These findings provide further insights into the structural details of the catalytic mechanism of the type III PKS enzymes.

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Structure-based engineering of benzalacetone synthase
Yoshihiko Shimokawa, Hiroyuki Morita, Ikuro Abe*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l ei n f o
Article history:
Received 17 June 2010
Revised 4 July 2010
Accepted 7 July 2010
Available online 11 July 2010
Keywords:
Polyketide synthase
Benzalacetone synthase
Enzyme
Site-directed mutagenesis
Enzyme engineering
a b s t r a c t
Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide syn-
thases (PKSs), sharing 70% amino acid sequence identity and highly homologous overall protein struc-
tures. BAS catalyzes the decarboxylative coupling of 4-coumaroyl-CoA with malonyl-CoA to produce
the diketide benzalacetone, whereas CHS produces the tetraketide chalcone by iterative condensations
with three molecules of malonyl-CoA, and folding the resulting intermediate into a new aromatic ring
system. Recent crystallographic analyses of Rheum palmatum BAS revealed that the characteristic substi-
tution of Thr132 (numbering of Medicago sativa CHS2), a conserved CHS residue lining the active-site cav-
ity, with Leu causes steric contraction of the BAS active-site to produce the diketide, instead of the
tetraketide. To test this hypothesis, we constructed a set of R. palmatum BAS site-directed mutants
(L132G, L132A, L132S, L132C, L132T, L132F, L132Y, L132W and L132P), and investigated the mechanistic
consequences of the point mutations. As a result, the single amino acid substitution L132T restored the
chalcone-forming activity in BAS, whereas the Ala, Ser, and Cys substitutions expanded the product chain
length to produce 4-coumaroyltriacetic acid lactone (CTAL) after three condensations with malonyl-CoA,
but without the formation of the aromatic ring system. Homology modeling suggested that this is prob-
ably caused by the restoration of the ‘coumaroyl binding pocket’ in the active-site cavity. These findings
provide further insights into the structural details of the catalytic mechanism of the type III PKS enzymes.
? 2010 Elsevier Ltd. All rights reserved.
Benzalacetone synthase (BAS), a member of the plant-specific
chalcone synthase (CHS) superfamily of type III polyketide
synthases (PKSs),1–3produces the diketide benzalacetone by the
condensation of 4-coumaroyl-CoA with one molecule of malonyl-
CoA (Figs. 1 and 2).4–7BAS generates the C6–C4scaffold of the
biologically active phenylbutanoids, including raspberry ketone
in raspberry fruit and the anti-inflammatory glucoside lindleyin
in rhubarb.4In contrast, CHS, which shares ca. 70% amino acid
sequence identity with BAS, catalyzes iterative condensations of
4-coumaroyl-CoA with three molecules of malonyl-CoA, and folds
the resulting tetraketide intermediate into a new aromatic ring
system to produce naringenin chalcone (Figs. 1 and 2).1–3Recent
crystallographic and site-directed mutagenesis studies have begun
to reveal that the functional diversity of the CHS-superfamily type
III PKSs principally arises from simple steric modulations of the ac-
tive-site architecture.8–18
We previously reported the cloning and characterization of BAS
from rhubarb (Rheum palmatum),4–6and solved the X-ray crystal
structures of both the wild-type and chalcone-producing I214L/
L215F mutant (numbering of Medicago sativa CHS2).7The crystal
structures at 1.8 Å resolution revealed that BAS utilizes an alterna-
tive, novel active-site pocket to bind the aromatic moiety of the
coumarate, instead of CHS’s ‘coumaroyl binding pocket’, which is
lost in the active-site of the wild-type BAS and restored in the chal-
cone-producing double mutant (Fig. 3a and c).7In addition to the
residues Ile214/Leu215, which are located at the junction between
the active-site cavity and the CoA binding tunnel, the crystal struc-
tures suggested that the active-site residue Leu132, corresponding
to Thr132 in CHS, at the entrance of the ‘coumaroyl binding pock-
et’, also plays an important role in the benzalacetone-forming
activity. To test this hypothesis, and to clarify the structure–func-
tion relationships of the type III PKS enzymes, we constructed a
set of site-directed mutants of R. palmatum BAS (L132G, L132A,
L132S, L132C, L132T, L132F, L132Y, L132W and L132P), and inves-
tigated the mechanistic consequences of the point mutations.
The point mutants were expressed in Escherichia coli as GST-
tagged fusion proteins, at levels comparable to that of the wild-
type enzyme.19The recombinant enzymes were then purified to
homogeneity by Glutathione Sepharose 4B affinity column, and
the GST-tags were removed by digestion with PreScission Prote-
ase.20All of the mutants maintained the benzalacetone-forming
activities at pH 8.0, whereas at pH 6.5, the point mutants afforded
the triketide pyrone bisnoryangonin (BNY), after two condensa-
tions with malonyl-CoA, as in the case of the wild-type enzyme
(Fig. 4).21,22It should be noted that, as previously reported, R. pal-
matum BAS shows pH dependency of the enzyme activity; the
0960-894X/$ - see front matter ? 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.022
Abbreviations: BAS, benzalacetone synthase; CHS, chalcone synthase; PKS,
polyketide synthase; STS, stilbene synthase; BNY, bisnoryangonin; CTAL, 4-
coumaroyltriacetic acid lactone.
* Corresponding author. Tel.: +81 3 5841 4740; fax: +81 3 5841 4744.
E-mail address: abei@mol.f.u-tokyo.ac.jp (I. Abe).
Bioorganic & Medicinal Chemistry Letters 20 (2010) 5099–5103
Contents lists available at ScienceDirect
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journal homepage: www.elsevier.com/locate/bmcl

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OH
O
CoAS
OH
Enz-S
OO
OH
H3C
O
Enz-S
OOO
OH
OOO-
OH
Enz-S
O
OH
O
OH
O
OH
O
OO
OH
OH
OO
OS-Enz
O
OH
O OH
HOOH
+ 1 x malonyl-CoA
+ 1 x malonyl-CoA
+ 1 x malonyl-CoA
4-coumaroyl-CoA
benzalacetone
bisnoryangonin (BNY)
4-coumaroyltriacetic acid lactone
(CTAL)
naringenin chalcone
Figure 1. Proposed mechanism for the formation of benzalacetone, bisnoryangonin (BNY), 4-coumaroyltriacetic acid lactone (CTAL), naringenin chalcone and resveratrol
from 4-coumaroyl-CoA and malonyl-CoA.
Figure 2. Comparison of the primary sequences of BAS and other type III PKSs. The secondary structures, a-helices (rectangles), b-strands (arrows), and loops (bold lines), of
BAS are also displayed. The catalytic triad Cys-His-Asn, and the residues considered to be crucial for the functional diversity of the type III PKSs are marked with * and #,
respectively. Abbreviations (GenBank accession numbers): R.pa.BAS, Rheum palmatum BAS (AAK82824); M.sa.CHS, M. sativa CHS (P30074); P.sy. STS, Pinus sylvestris STS
(AAB24341); H.an. BPS, Hypericum androsaemum BPS (AAL79808).
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benzalacetone-forming activity is maximum within the pH 8.0–8.5
range,whereasunderacidicconditions,BNYformationisdominant,
and it is obtained almost as a single product at pH 6.5.5Remarkably,
only the L132T mutant, in which Leu132 was substituted with Thr,
as in the case of CHS, restored the chalcone-forming activity, to pro-
duce naringenin chalcone after three condensations with malonyl-
CoA and the formation of the aromatic ring system. The L132T mu-
tantalsoproducedthetetraketidepyrone4-coumaroyltriaceticacid
lactone (CTAL) after three condensations with malonyl-CoA, along
with BNY and benzalacetone (Fig. 4). The yields of benzalacetone,
BNY, CTAL, and naringenin chalcone were 2.3%, 7.4%, 1.0%, and
0.5%, respectively. On the other hand, except for the L132G mutant,
large-to-small substitutions with Ala, Ser, and Cys expanded the
product chain length to yield CTAL after three condensations with
malonyl-CoA,butwithouttheformationofthearomaticringsystem
(Fig. 4). It was thus confirmed that the residue 132, located at the
entrance of the ‘coumaroyl binding pocket’, indeed plays a critical
role in the diketide formation reaction, along with the previously
reported active-site residues Leu215 and Ser338.5,6This is the first
demonstration that a single amino acid replacement restored the
chalcone-forming activity in R. palmatum BAS.
A steady-state kinetics analysis revealed that the BAS L132T mu-
tantshowedaKM= 4.1 lManda kcat= 1.60 ? 10?3min?1for4-cou-
maroyl-CoA, with respect to the chalcone-forming activity, with a
pH optimum of 6.5.23On the other hand, the previously reported
chalcone-forming BAS I214L/L215F double mutant exhibited a
KM= 33.5 lM and a kcat= 1.69 ? 10?1min?1for 4-coumaroyl-CoA,
representing a 13-fold increase in the kcat/KM value,5whereas
the BAS wild-type enzyme showed a KM= 10.0 lM and a kcat=
1.79 min?1for 4-coumaroyl-CoA, with respect to the benzalace-
tone-forming activity. It is remarkable that the KMvalue remained
atasimilarlevelinthepointmutant,despitethesignificantdecrease
of the kcatvalue. This suggests that the single amino acid replace-
ment mainly affects the catalytic process, rather than the substrate
binding to the active-site of the enzyme. It should be noted that
when the two mutations, L132T and I214L/L215F, were combined,
a BAS b CHS
d BAS L132Te BAS L132W
L132
T132
T132
L132
W132
C164
C164
C164
C164
C164
H303
H303
H303
H303
H303
S338
S338S338
S338
S338
N336
N336
N336
N336
N336
C197
C197
T197
C197
C197
L215
L215
L215
F215F215
I214
I214
I214
L214
L214
F265
F265
F265
F265
F265
c BAS I214L/L215F
Figure 3. Comparison of the active-site architecture of (a) wild-type BAS, (b) M. sativa CHS, (c) the BAS I214L/L215F mutant, (d) the BAS L132T mutant, and (e) the BAS L132W
mutant. The active-sites are represented by surface models. The coumarate that covalently binds to the active-site Cys in BAS and the naringenin molecules are shown as
black and green stick models, respectively. The bottoms of the ‘coumaroyl binding pocket’ are highlighted in purple.
Figure 4. Production of benzalacetone (at pH 8.0), BNY (at pH 6.5), CTAL (at pH 6.5) and naringenin chalcone (at pH 6.5) by wild-type BAS and its mutants.
Y. Shimokawa et al./Bioorg. Med. Chem. Lett. 20 (2010) 5099–5103
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the L132T/I214L/L215F triple mutation did not improve the chal-
cone-forming activity, but instead resulted in a significant loss of
activity (Fig. 4).19
In addition, the chalcone-forming L132T mutant showed broad
substrate specificity.24The enzyme also accepted benzoyl-CoA as
the starter substrate to produce a trace amount of 2,4,6-trihydr-
oxybenzophenone, after condensations of benzoyl-CoA with three
molecules of malonyl-CoA, along with benzoate-primed triketide
and tetraketide pyrones as major products, as in the case of the
previously reported CHS from Scutellaria baicalensis.24On the other
hand, when phenylacetyl-CoA was used as the starter substrate,
the mutant produced only phenylacetate-primed triketide and tet-
raketide pyrones. In contrast, the wild-type BAS produced only tri-
ketide pyrones from benzoyl-CoA and phenylacetyl-CoA starter
substrates. These observations suggest that the active-site cavity
of the BAS L132T mutant is large enough to accommodate the tet-
raketide products as in the case of CHS.
Interestingly, the replacement of Leu132 with bulky aromatic
residues, Phe, Tyr and Trp, caused a 1.2-fold increase in the benz-
alacetone-forming activity at pH 8.0, whereas the BNY-forming
activity was retained or significantly decreased at pH 6.5 (Fig. 4).
These point mutations thus resulted in increased product specific-
ity for the diketide benzalacetone formation, in sharp contrast to
the aforementioned large-to-small substitutions. On the other
hand, it was quite surprising that the substitution of Leu132 with
the small Gly residue did not significantly affect the enzyme activ-
ity (Fig. 4). In the crystal structure of the wild-type BAS, Leu132 is
located within a loop region. It is tempting to speculate that the
Leu to Gly substitution may retain the flexibility of the loop, there-
by maintaining a similar active-site structure to that of the wild-
type enzyme. In contrast, when Leu132 was substituted with the
cyclized and less flexible Pro, the L132P mutant exhibited drasti-
callydecreased benzalacetone-
(Fig. 4). This result also suggests that the flexibility of the loop
structure is important for the enzyme activity.
To further clarify the structure-function relationship, homology
models of the L132T and L132W mutants were constructed on the
basis of the crystal structure of the wild-type R. palmatum BAS
(PDB code: 3A5Q),25and their active-site architectures were com-
pared with those of the wild-type and the chalcone-producing
I214L/L215F double mutant of BAS7, and the Medicago sativa
CHS8(Fig. 3). The homology models predicted that the L132T mu-
tant restored the ‘coumaroyl binding pocket’ in the active-site cav-
ity (Fig. 3d). The total cavity volume of the L132T mutant is
estimated to be 400 Å3, which is slightly larger than that of the
wild-type (350 Å3) and almost as large as that of the I214L/L215F
mutant (400 Å3), but much smaller than that of the M. sativa CHS
(750 Å3). As previously proposed by Noel and Schröder, Thr132 in
CHS forms a hydrogen bond with the neighboring Glu192, and is
thought to control the folding of the tetraketide intermediate so
that the Claisen-type cyclization produces the chalcone scaffold.10
These observations suggest that the L132T substitution opens a
gate to the buried ‘coumaroyl-binding pocket’, thereby increasing
the polyketide chain elongation by up to three condensations with
malonyl-CoA, and leading to the formation of chalcone. On the
other hand, in the L132W mutant, the replacement of Leu132 with
the bulky Trp further blocks the entrance of the pocket. In this
case, the total cavity volume is estimated to be 300 Å3, which is
smaller than that of the wild-type enzyme (350 Å3), resulting in
the interruption BNY formation, and instead improving the speci-
ficity for the benzalacetone-forming activity (Fig. 3e).
In summary, we investigated the functional role of the active-
site residue Leu132 of R. palmatum BAS, on the basis of the X-ray
crystal structures. We found that a single amino acid substitution,
L132T, restored the chalcone-forming activity of the enzyme.
Homology modeling suggested that this is probably caused by
andBNY-formingactivities
the restoration of the ‘coumaroyl binding pocket’ in the active-site
cavity. These findings provide further insights into the structural
details of the catalytic mechanisms of the type III PKS enzymes.
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science
and Technology, Japan (to I.A. and H.M.), and grants from The Naito
Foundation (to I.A.) and Takeda Science Foundation (to H.M.).
References and notes
1. Abe, I.; Morita, H. Nat. Prod. Rep. 2010, 27, 809.
2. Austin, M. B.; Noel, J. P. Nat. Prod. Rep. 2003, 20, 79.
3. Schröder, J. In Comprehensive Natural Products Chemistry; Elsevier: Oxford,
1999; Vol. 1. p 749.
4. Abe, I.; Takahashi, Y.; Morita, H.; Noguchi, H. Eur. J. Biochem. 2001, 268,
3354.
5. Abe, I.; Sano, Y.; Takahashi, Y.; Noguchi, H. J. Biol. Chem. 2003, 278, 25218.
6. Abe, T.; Morita, H.; Noma, H.; Kohno, T.; Noguchi, H.; Abe, I. Bioorg. Med. Chem.
Lett. 2007, 17, 3161.
7. Morita, H.; Shimokawa, Y.; Tanio, M.; Kato, R.; Noguchi, H.; Sugio, S.; Kohno, T.;
Abe, I. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 669.
8. Ferrer, J. L.; Jez, J. M.; Bowman, M. E.; Dixon, R. A.; Noel, J. P. Nat. Struct. Biol.
1999, 6, 775.
9. Jez, J. M.; Austin, M. B.; Ferrer, J.; Bowman, M. E.; Schröder, J.; Noel, J. P. Chem.
Biol. 2000, 7, 919.
10. Austin, M. B.; Bowman, M. E.; Ferrer, J. L.; Schröder, J.; Noel, J. P. Chem. Biol.
2004, 11, 1179.
11. Austin, M. B.; Izumikawa, M.; Bowman, M. E.; Udwary, D. W.; Ferrer, J. L.;
Moore, B. S.; Noel, J. P. J. Biol. Chem. 2004, 279, 45162.
12. Sankaranarayanan, R.; Saxena, P.; Marathe, U. B.; Gokhale, R. S.; Shanmugam,
V. M.; Rukmini, R. Nat. Struct. Mol. Biol. 2004, 11, 894.
13. Morita, H.; Kondo, S.; Oguro, S.; Noguchi, H.; Sugio, S.; Abe, I.; Kohno, T. Chem.
Biol. 2007, 14, 359.
14. Shomura, Y.; Torayama, I.; Suh, D. Y.; Xiang, T.; Kita, A.; Sankawa, U.; Miki, K.
Proteins 2005, 60, 803.
15. Goyal, A.; Saxena, P.; Rahman, A.; Singh, P. K.; Kasbekar, D. P.; Gokhale, R. S.;
Sankaranarayanan, R. J. Struct. Biol. 2008, 162, 411.
16. Rubin-Pitel, S. B.; Zhang, H.; Vu, T.; Brunzelle, J. S.; Zhao, H.; Nair, S. K. Chem.
Biol. 2008, 15, 1079.
17. Jez, J. M.; Ferrer, J. L.; Bowman, M. E.; Dixon, R. A.; Noel, J. P. Biochemistry 2000,
39, 890.
18. Abe, I.; Watanabe, T.; Morita, H.; Kohno, T.; Noguchi, H. Org. Lett. 2006, 8, 499.
19. Site-directed mutagenesis: The plasmids expressing the BAS mutants (L132G,
L132A, L132T, L132S, L132C, L132F, L132Y, L132W, and L132P) were
constructed with a QuikChange Site-Directed Mutagenesis Kit (Stratagene),
according to the manufacturer’s protocol, using the following pairs of primers
(mutated codons are underlined): L132G (50-CTCATCGTGTGTTGCGGAGC
CGGCGTTGAC-30and 50-GTCAACGCCGGCTCCGCAACACACGA-30), L132A (50-CT
CATCGTGTGTTGCGCAGCCGGCGTTGAC-30and 50-CATGTCAACGCCGGCTGCGCA
ACACACGAT-30), L132T (50-CTCATCGTGTGTTGCACAGCCGGCGTTGAC-30and 50-
CATGTC AACGCCGGCTGTGCAACACACGAT-30), L132S (50-CTCATCGTGTGTTGC
TCAGCCGGCGTTGAC-30and 50-GTCAACGCCGGCTGAGCAACACACGATGAG-30),
L132C (50-CTCATCGTGTGTTGCTGTGCCGGCGTTGAC-30and 50-GTCAAC GCCGG
CACAGCAACACACGATGAG-30),L132F
TTGAC-30and 50-GTCAACGCCGGCAAAGCAACACACGATGAG-30), L132Y (50-CTC
ATCGTGTGTTGCTATGCCGGCGTTGAC-30and 50-GTCAAC GCCGGCATAGCAACA
CACGATGAG-30), L132W (50-CTCATCGTGTGTTGCTGGGCCGGCGTTGAC-30and
50-GTCAACGCCGGCCCAGCAACACACGATGAG-30), and L132P (50-CTCATCGTG
TGTTGCCCAGCCGGCGTTGAC-30and 50-GTCAACGCCGGCTGGGCAACACACGAT
GAG-30). The expression plasmid for the L132T/I214L/L215F triple mutant was
also constructed in a similar manner.
20. Expression and purification: After confirmation of the sequence, the plasmid was
transformed into E. coli M15. The cells harboring the plasmid were cultured to
an A600 of 0.6 in LB medium containing 100 lg/mL of ampicillin at 37 ?C.
Subsequently, 1.0 mM isopropylthio-b-D-galactopyranoside was added to
induce protein expression, and the cells were further cultured at 23 ?C for
16 h. All of the following procedures were performed at 4 ?C. The E. coli cells
were harvested by centrifugation at 5000g, and were resuspended in 50 mM
Tris–HCl buffer (pH 8.0), containing 0.2 M NaCl, 5% (v/v) glycerol and 2 mM
DTT (buffer A). The cells were disrupted by sonication, and the lysate was
centrifuged at 12,000g for 30 min. The supernatant was loaded onto a
Glutathione Sepharose 4B affinity column (GE Healthcare) equilibrated with
buffer A, and the column was then washed with buffer A. The GST-tag was
cleaved on the column by PreScission Protease (GE Healthcare) overnight, and
then the recombinant BAS mutant was eluted with buffer A. The resultant BAS
protein thus contains three additional residues (Gly-Pro-Gly) at the N-terminal
flanking region, derived from the PreScission Protease recognition sequence.
The protein concentration was determined by the Bradford method (Protein
Assay, Bio-Rad) with bovine serum albumin as the standard.
(50-CTCATCGTGTGTTGCTTTGCCGGCG
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21. Enzyme reaction: The reaction mixture contained 54 lM of 4-coumaroyl-CoA
(benzoyl-CoA or phenylacethyl-CoA), 108 lM of malonyl-CoA, and 20 lg of the
purified enzyme in a final volume of 500 lL of 100 mM KPB buffer (pH 6.5) or
Tris–HCl buffer (pH 8.0), containing 1 mM EDTA. Incubations were performed
at 30 ?C for 20 min and were stopped by the addition of 50 lL of 20% HCl. The
products were then extracted with 3 mL of ethyl acetate. The products were
separated by reverse-phase HPLC (JASCO 880) on a TSK-gel ODS-80Ts column
(4.6 ? 150 mm, TOSOH), at a flow rate of 0.8 mL/min. Gradient elution was
performed with H2O and MeOH, both containing 0.1% TFA: 0–5 min, 30%
MeOH; 5–17 min, linear gradient from 30% to 60% MeOH; 17–25 min, 60%
MeOH; 25–27 min, linear gradient from 60% to 70% MeOH. Elutions were
monitored by a multichannel UV detector (MULTI 340, JASCO) at 280; UV
spectra (198–400 nm) were recorded every 0.4 s. Online LC–ESIMS spectra
were measured with an Agilent Technologies HPLC 1100 series HPLC coupled
to a Bruker Daltonics Esquire4000 ion trap mass spectrometer fitted with an
ESI source. HPLC separations were performed under the same conditions as
described above. The ESI capillary temperature and the capillary voltage were
320 ?C and 4.0 V, respectively. The tube lens offset was set at 20.0 V. All spectra
were obtained in the positive mode, over a mass range of m/z 50–500, and at a
range of one scan every 0.2 s. The collision gas was helium, and the relative
collision energy scale was set at 30.0% (1.5 eV).
22. 4-Coumaroyl-CoA was chemically synthesized as described: Stöckigt, J.; Zenk,
M. H. Z. Naturforsch., C: Biosci. 1975, 30, 352 [2-14C]Malonyl-CoA was purchased
from GE Healthcare. Benzoyl-CoA, phenylacetyl-CoA and malonyl-CoA were
purchased from Sigma.
23. Enzyme kinetics: Steady-state kinetic parameters were determined by using
[2-14C]malonyl-CoA (1.8 mCi/mmol) as the substrate. The experiments were
performed in triplicate using four concentrations of 4-coumaroyl-CoA (27.0,
13.5, 6.8, and 3.4 lM) in the assay mixture, containing 108 lM of malonyl-CoA,
20 lg of purified enzyme, and 1 mM EDTA, in a final volume of 500 lL of
100 mM potassium phosphate buffer, pH6.5. The reactions were incubated at
30 ?C for 20 min. The reaction products were extracted and separated by TLC
(Merck Art. 1.11798 Silica Gel 60 F254; ethyl acetate/hexane/AcOH = 63:27:5,
v/v/v). Radioactivities were quantified by autoradiography using a bioimaging
analyzer BAS-2000II (FUJIFILM). Lineweaver–Burk plots of data were employed
to derive the apparent KMand kcatvalues (average of triplicates), using the
EnzFitter software (BIOSOFT).
24. Morita, H.; Takahashi, Y.; Noguchi, H.; Abe, I. Biochem. Biophys. Res. Commun.
2000, 279, 190.
25. Homology modeling: The models of the BAS L132T and L132W were generated
by the SWISS-MODEL package (http://expasy.ch/spdbv/) provided by the Swiss-
PDB-Viewer program (Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714)
based on the crystal structure of wild-type BAS (PDB code: 3A5Q). The model
quality was assessed using PROCHECK (Liang, J.; Edelsbrunner, H.; Woodward,
C. Protein Sci. 1998, 7, 1884). In the Ramachandran plot calculated for the
model, most of the amino acid residues were present in the energetically
allowed regions with only a few exceptions, primarily Gly residues that can
adopt phi/psi angles in all four quadrants. The cavity volume was calculated by
the program CASTP (http://cast.engr.uic.edu/cast/). All protein structure figures
were prepared with PYMOL (DeLano Scientific, http://www.pymol.org).
Y. Shimokawa et al./Bioorg. Med. Chem. Lett. 20 (2010) 5099–5103
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