1
Engineered biosynthesis of β
-
alkyl tryptophan analogs
Christina E. Bo
ville
+
, Remkes A. Scheele
+
, Philipp Koch, Sabine
Brinkmann
-
Chen, and Andrew R.
Buller*
, Frances H.
Arnold*
Abstract:
Non
-
canonical amino acids (ncAAs) with dual stereocenters at the α and β positions are valuable precursors to natural products
and therapeutics. Despite the potential applications of such
bioactive β
-
branched ncAAs
, their availability is limited due to the inefficiency of
the multi
-
step methods used to prepare them.
Here we report a stereoselective biocatalytic synthesis of β
-
branched tryptophan analogs using
an engineered variant of
Pyrococcus furiosus
tryptophan synthase (
Pf
TrpB),
Pf
TrpB
7E6
.
Pf
TrpB
7E6
is the first biocatalyst
to
synthesize bulky β
-
branched tryptophan analogs
in a single step
,
with demonstrated
access to 27 ncAAs. The molecular basis for the efficient catalysis and
br
oad
substrate
tolerance
of
Pf
TrpB
7E6
was explored through X
-
ray crystallography and UV
-
visible light spectroscopy, which revealed that a
combination of active
-
sit
e and remote mutations increase
the abundance and persistence of a key reactive intermediate.
Pf
TrpB
7E6
provides
an operationally simple and environmentally benign platform for preparation of β
-
branched tryptophan building blocks.
2
Table
of Contents
Results and Discussion
...
...............................................................................................................
.
............................................................
....
2
Table
S1.
Summary of the mutations (green) included in the recombination library that led to
Pf
TrpB
7E6
............................................
..............
.............................
....
.2
Table
S2.
Thermostability of evolve
d PfTrpB variants...........................................
..........................................................................................
..
........2
Table
S3.
Enzymatic formation of α
-
keto acids.....................
......................................
.............................................
.
.......................................
...
......2
Table
S4.
Isosbestic point of tryptophan analogs and the corresponding indole analogs.
......................................................................
.............................
..........
...
....3
Table
S
5
.
Tryptophan analogs synthesized in pr
eparative reactions
.
................................................................................................
...
............................................
...
.4
Table
S6.
Optimization of reaction yields.
..................................................................
..............................
............................................................................................
..
5
Figure S1.
Locations of mutations in
Pf
TrpB
7E
6
..................
......................................
..............................
.............................................................................................
..
6
Figure S2.
Characterization of
Pf
TrpB
7E6
.
............................................................................
..............................
..................................................................................
..
6
Figure S3.
Substrate binding and conformational changes in
Pf
Trp
B
with iPrSer.
............................................
.............................
..................................................
...
.7
Experimental Procedures
.............................................................................................................................
......
.......
........................................................................
..
7
Table
S7.
Primers for random mutagenesis.
.
.............................................................................................................................
......
.........
.........................................
...
8
Table
S8.
Summary of the
mutations
that were subjected to recombination
and their variant
-
of
-
origin................................................................................
....
..........
..
8
Table
S9.
Primers for cloning recombination libraries.
.......................................
................................
..
..............................................................................................
...
8
Table
S10.
Primers for site
-
directed and site
-
saturation mutagenesis.
......................................................
...........................................................................................8
Table
S
11.
Crystallographic data collection and refinement statistics.
..................................................................................................................
..............................14
Product Characterization
...........................................................
..........................................................................................................................
..............................15
Results and Discussion
Table
S1.
Summary of the mutations (green) included in the recombination libra
ry that led to
Pf
TrpB
7E6
.
Substrate
Serine
Threonine
β
-
EtSer
Variant
Pf
TrpB
2G9
Pf
TrpB
4D11
Pf
TrpB
4G1
Pf
TrpB
2B9
Pf
TrpB
2B9
L161A
Pf
TrpB
0E3
Pf
TrpB
8C8
Mutations
T292S
E17G
I68V
F274S
T321A
F95L
I16V
V384
A
L161A
L91P
V173E
Table
S2
.
T
hermostability of evolved
PfTrpB variants. Thermostability is reported as the temperature at which half the activity is lost (T
50
) after 1
-
hour incubation.
Pf
TrpB Variant
T
50
(°C)
Pf
TrpB
2B9
95.0 ± 0.2
Pf
TrpB
2B9
L161A
81.3 ± 0.7
Pf
TrpB
0E3
86.0 ± 0.1
Pf
TrpB
8C8
89.3 ± 0.8
Pf
TrpB
7E6
86.6 ± 0.1
Table
S3
.
Enzymatic formation of α
-
keto acids. Change in absorption at 320
nm was monitored for 10 min
. Deamination rate
in units
of
sec
-
1
is
calculated using
the extinction coefficient of
α
-
ketobutyrate (
20
M
-
1
cm
-
1
). N.D
. no
t detected
,
E(A
-
A) was not observed under these reaction conditions.
Substrate deamination (
sec
-
1
)
Enzyme
Thr
β
-
EtSer
β
-
PrSer
Pf
TrpB
2B9
0.4
N.D
.
N.D
.
Pf
TrpB
8C8
0.
1
0.
2
0.
2
Pf
TrpB
7E6
0.
1
0.
2
0.1
3
Table
S4
.
Isosbestic point of tryptophan analogs and the
corresponding indole analog
s
.
Nucleophile substrate
Isosbestic point (nm)
Indole
277
2
-
methylindole
279
4
-
methylindole
279
4
-
fluoroindole
267
5
-
methylindole
280
5
-
fluoroindole
282
5
-
chloroindole
260
6
-
methylindole
273
7
-
methylindole
272
Indazole
276
7
-
azaindole
292
4
Table
S5.
Tryptophan analogs synthesized in preparative reactions. Preparative reactions were performed with 100 μmol of nucleophile an
d two equivalents of electrophile with varied loading of
Pf
TrpB
2G8
. TTN are
reported with yields in parenthesis
Nucleophilic Substrate
Indole
2
-
methylindole
4
-
methylindole
4
-
fluoroindole
5
-
methylindole
5
-
fluoroindole
5
-
chloroindole
6
-
methylindole
7
-
methylindole
7
-
azaindole
Electrophilic Substrate
Thr
5400 (72%)
a
3700 (92%)
c
1600 (47%)
c
3600 (87%)
c
1800 (45%)
c
3200 (91%)
c
100 (20%)
h
1200 (78%)
f
3200 (63%)
b
3900 (77%)
b
β
-
EtSer
5300 (88%)
b
2800 (94%)
c
600 (30%)
e
1800 (89%)
e
100 (20%)
h
2900 (97%)
c
-
500 (35%)
f
1900 (97%)
e
-
.
β
-
PrSer
1900 (77%)
d
200 (21%)
g
100
(23%)
h
200 (39%)
h
20 (7%)
i
400 (44%)
g
-
20 (10%)
i
1100 (56%)
e
-
.
Catalyst loading (%):
a
0.01%;
b
0.02%;
c
0.03%;
d
0.04%;
e
0.05%;
f
0.07%;
g
0.1%;
h
0.2%;
i
0.4%;
-
= not tested
5
Table
S6
.
Optimization of reaction yields. Reaction yields can be
improved by increasing the equivalents of electrophilic substrate or increasing catalyst loading.
LCMS reactions with PfTrpB2B9 and PfTrpB7E6 were conducted with 20 mM indole, 1 or 10 equivalents of electrophilic substrate,
and varied catalyst loading
(0.0
1%
–
0.1%). Reactions were incubated for 24
h
at 75 ºC and analyzed by LCMS.
Enzyme
Catalyst Loading (%)
Product
Electrophilic Substrate Equivalents
HPLC yield (%)
Pf
TrpB
2B9
0.01
β
-
MeTrp
1
13
0.01
β
-
MeTrp
10
24
Pf
TrpB
7E6
0.01
β
-
MeTrp
1
48
0.01
β
-
MeTrp
10
97
0.05
β
-
MeTrp
1
95
0.1
β
-
MeTrp
1
95
0.01
β
-
EtTrp
1
46
0.01
β
-
EtTrp
10
62
0.05
β
-
EtTrp
1
91
0.1
β
-
EtTrp
1
96
0.01
β
-
PrTrp
1
18
0.01
β
-
PrTrp
10
14
0.05
β
-
PrTrp
1
52
0.1
β
-
PrTrp
1
59
6
Figure S1.
Locations of mutations
in
Pf
TrpB
7E
6
.
Pf
TrpB
7E6
contains nine mutations relative to wild
-
type
Pf
TrpB. Mutations are distributed throughout the enzyme
and are indicated in red.
L161A is an active site mutation.
Figure S2.
Characterization of
Pf
TrpB
7E6
. (a) Comparison of TTN values with
Pf
TrpB
7E6
and
Pf
TrpB
7E6
L161V
. (b) Comparison of
Pf
TrpB
7E6
TTN with serine and
indole.
Bars represent the average of all data points, with individual reactions shown as circles. At minimum, reactions were perform
ed in du
plicate.
7
Figure S3.
Substrate binding and conformational changes in
Pf
TrpB
with iPrSer.
(a)
(2
S
, 3
S
)
-
β
-
iPrSer is bound to
Pf
TrpB
7E6
as E(Aex
1
) (PDB: 6CUT, purple). The
Pf
TrpB
7E6
COMM domain assumes a more closed conformation when compared to wild
-
type
Pf
TrpB
without substrate
(PDB: 5D
VZ
, gray
)
or with Ser as E(Aex
1
)
(PDB 5DW0, lime)
.
(b)
iPrSer shown
with F
o
-
F
c
map contoured at 2.5
σ
(green).
Hydrogen bonds are shown as red dashes.
Experimental Procedures
General experimental methods.
Chemicals and
reagents were purchased from commercial sources and used without
further purification. Proton and carbon NMR spectra were recorded on a Bruker 400 MHz (100 MHz) spectrometer equipped with a
cryogenic probe. Proton chemical shifts are reported in ppm (δ) re
lative to tetramethylsilane and calibrated using the residual solvent
resonance (DMSO, δ 2.50 ppm). Data are reported as follows: chemical shift (multiplicity [singlet (s), doublet (d), doublet o
f doublets
(dd), doublet of doublets of doublets (ddd), tripl
et (t), triplet of doubles (td), multiplet (m)], coupling constants [Hz], integration).
Carbon NMR spectra were recorded with complete proton decoupling. Carbon chemical shifts are reported in ppm relative to
tetramethylsilane and calibrated using the resi
dual solvent proton resonance as an absolute reference. All NMR spectra were
recorded at ambient temperature (about 25 °C). Preparative reversed
-
phase chromatography was performed on a Biotage Isolera
One purification system, using C
-
18 silica as the stati
onary phase, with CH
3
OH as the strong solvent and H
2
O (0.1% HCl by weight)
as the weak solvent. Liquid chromatography/mass spectrometry (LCMS) was performed on an Agilent 1290 UPLC
-
LCMS equipped
with a C
-
18 silica column (1.8 μm, 2.1 × 50 mm) using CH
3
CN/H
2
O (0.1% acetic acid by volume): 5% to 95% CH
3
CN over 4 min; 1
mL/min.
Cloning.
Pf
TrpB
WT
(UNIPROT ID Q8U093) was previously codon optimized for expression in
Escherichia coli
, and cloned
into pET
-
22b(+) with a C
-
terminal 6x His tag
.
[1]
Parent variant
Pf
TrpB
2B9
(E17G, I68V, T292
S, F274S, T321A, F95L, I16V, V384A)
was cloned and expressed as described previously
.
[2]
Substrate modeling in the active site.
β
-
EtSer was modeled as E(A
-
A) in the crystal structure of
Pf
TrpB
2B9
(PDB: 5VM5)
by
following the coordinates of the
E(A
-
A)
formed from Ser and
following the known
E
-
stereochemistry of the intermediate
.
E
ach
staggered rotamer
for the methyl group
was sampled manually in Coot
. T
he single rotamer lacking a steric clash was
selected and
the entire active site
subject to 10
cycles
of structure idealization in Refmac5.
Construction of random mutagenesis libraries.
Random mutagenesis libraries were generated with the appropriate
Pf
TrpB gene as template by the addition of 200
–
400 μM MnCl
2
to a
Taq
PCR reaction as reported previously
.
[1]
PCR fragments were
treated with DpnI for 2 h at 37 ºC, purified by gel extraction, and then inserted into a pET
-
22b(+) vector via Gibson assembly
.
[3]
BL
21(DE3) E. cloni
®
Express cells were transformed with the Gibson assembly product.
8
Table
S7.
Primers for random mutagenesis.
Primer
Sequence (5’ to 3’)
Random mutagenesis forward (NdeI)
GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG
Random mutagenesis
reverse (XhoI)
GCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAG
pET22
-
b(+) Forward
CATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTC
pET22
-
b(+) Reverse
CTCGAGCACCACCACCACCACCACTGAGATCCGGC
Construction of recombination libraries.
Recombination libraries used primers with
degenerate codons to cause an
equal ratio of mutant and wild
-
type residues at a given site (I16V, E17G, I68V, V173E, F274S/L, T321A, and V384A). The library was
prepared in two rounds of PCR. For the first round, a PCR with Phusion
®
polymerase produced four fragments of the PfTrpB
8C8
gene
(NdeI to I16/E17, I16/E17 to V173, V173 to T321, T321 to XhoI). Fragments were treated with DpnI for one hour at 37 °C and pu
rified
by a preparative agarose gel. The individual fragments were used a
s template in an assembly PCR with pET22
-
specific flanking
primers to generate the full
-
length insert. This assembled product was then used as template for the second round of PCR
amplification, producing another four fragments of the PfTrpB
8C8
gene (NdeI
to I68, I68 to F274, F274 to V384, V384 to XhoI). The
fragments were treated as described above. The complete library was then inserted into pET
-
22b(+) via Gibson assembly.
[
3]
BL21(DE3) E. cloni
®
Express cells were transformed with the library.
Table
S
8.
Summary of the
mutations
that were subjected to recombination
and their variant
-
of
-
origin
.
Variant
Screened Substrate
Mutations
1
Pf
TrpB
4D11
Serine
E17G, I68V, F274S,
T321A
2
Pf
TrpB
2B9
Threonine
I16V, V384A
Pf
TrpB
8C8
β
-
EtSer
V173E
Table
S
9.
Primers for cloning recombination libraries.
Fragment
Forward primer (5’ to 3’)
Reverse primer (5’ to 3’)
NdeI to I16/E17
GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG
TTCAGGGGTYCTAYCAGCGTTTCTGG
I16/E17 to V173
CCAGAAACGCTGRTAGRACCCCTGAA
TATTCAAAAGTAGCTWCCCAATCACGCAGAGCC
V173 to T321
GGCTCTGCGTGATTGGGWAGCTACTTTTGAATA
TTCTTCATCGGTTACTGYCACGTATTCAGCAC
T321 to XhoI
GTGCTGAATACGTGRCAGTAACCGATGAAGAA
GCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAG
NdeI to I68
GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG
CACGTTTCAGGTATAYTTTAGCACCACCG
I68 to F274
CGGTGGTGCTAAARTATACCTGAAACGTG
GACAGCATGCCATGMRACACACCAACCTGACC
F274 to V384
GGTCAGGTTGGTGTGTYKCATGGCATGCTGTC
GAGCACGTTGCC
AGATRCTTTCAGGACAATATC
V384 to XhoI
GATATTGTCCTGAAAGYATCTGGCAACGTGCTC
GCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAG
Site
-
directed and site
-
saturation mutagenesis.
Site
-
directed mutagenesis was performed with QuikChange
®
or Q5
®
kits
per manufacturer’s recommendations. Q5
®
primers were designed using the NEBASECHANGER
®
software. PCR with Phusion
®
polymerase was used to site
-
saturate L161 in
Pf
TrpB
2B9
. Primers were mixed as described previously.
[4]
Constructs were used to
transform BL21(DE3) E. cloni
®
Express cells.
Table
S10
.
Primers for site
-
directed a
nd site
-
saturation mutagenesis.
Target site
Forward primer (5’ to 3’)
Reverse primer (5’ to 3’)
Pf
TrpB
2B9
L161G
CCGGTTCTCGCACCGGGAAAGACGCAATCAACG
GGCCAAGAGCGTGCCCTTTCTGCGTTAGTTGC
Pf
TrpB
2B9
L161
site
-
saturation
CGTAATTCCAGTTAACTCCGGTTCTCGCACC
XXX
AAAGACGCAATCAACG
GGTGCGAGAACCGGAGTTAACTGGAATTACGTTT
GC
Pf
TrpB
7E6
A161V
TTCTCGCACCGTGAAAGACGCAA
CCGGAGTTAACTGGAATTACGTTTG
XXX in site saturation primers denotes NDT, VHG, or TGG.
[4]
Variant DNA Sequencing.
Variants identified through screening were DNA sequenced to determine their identities. The DNA
sequences of the
Pf
TrpB gene are included here. All variants were cloned into a pET22
-
b(+) vector as described above.
Pf
TrpB
2B9
L161A
ATGTGGTTCGGTGAATTTGGTGGTCA
GTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAATTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACTGCTGGCAAAG
CTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGCGAAAGACGCAATCAACGAGGCT
9
CTGCGTGATTGGGTGGCTACTTTTGAATACACCC
ACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAG
CCTGAACGCAGGTCAGGTTGGTGTGTCCCATGGCATGCTGTCCTACT
TTCTGCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGGCTGTAACCGATGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTAC
CGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTG
GCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGAT
CATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGTCCTGAAAGCGTCTGGCAACGTGCTCGAGCACCACCA
CCACCACCACTGA
Pf
TrpB
2B9
L161G
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGA
AGAATTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACTGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGT
GCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGGGAAAGACGCAATCAACGAGGCT
CTGCGTGATTGGGTGGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCG
GTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTCCCATGGCATGCTGTCCTACT
TTCTGCAGGACGAAGAAGGTCAGAT
CAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGGCTGTAACCGATGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTAC
CGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGAT
CATCATCGTAAACCTGTCTGGTCGTGGT
GACAAAGACCTGGATATTGTCCTGAAAGCGTCTGGCAACGTGCTCGAGCACCACCA
CCACCACCACTGA
Pf
TrpB
2B9
L161V
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAATTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACG
CCATCGGTCAGGCACTGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGTGAAAGACGCAATCAACGAGGCT
CTGCGTGA
TTGGGTGGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGTAAAGGC
CTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTCCCATGGCATGCTGTCCTACT
TTCTGCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGGCTGTAACCGATGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTAC
CGAAGGTATCATCC
CAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGAT
CATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGTCCTGAAAGCGTCTGGCAACGTGCTCGAGCACCACCA
CCACCACCACTGA
Pf
TrpB
0E3
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAAAGCTTA
CAAACGT
TTCAAAGATGACGAAGAATTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACCGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGC
GTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGCGAAAGACGCAATCAACGAGGCT
CTGCGTGATTGGGTGGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTCCCATGGCATGCTGTCCTACT
TTCT
GCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGGCTGTAACCGACGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTA
CCGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGA
TCATCATC
GTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGTCCTGAAAGCGTCTGGCAACGTGCTCGAGCACCACC
ACCACCACCACTGA
Pf
TrpB
8C8
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAGTTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCC
ACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACCGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAG
AAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGCGAAAGACGCAATCAACGAGGCT
CTGCGTGATTGGGAGGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCG
TTGCTT