SUPPORTING INFORMATION
1
Table
of Contents
Results and Discussion
...
...............................................................................................................
.
............................................................
....
1
T
able S1
.
Summary of the mutations (green) included in the recombination library that led to
Pf
TrpB
7E6
............................................
.......
.......
.............................
....
.1
Table S2
.
Thermostability of evolve
d PfTrpB variants...........................................
..........................................................................................
..
........1
Table S3
.
Engineering
Pf
TrpB
through directed
evolution for improved β
-
EtTrp
production
.
.................................................................................
...............................1
Table S4
.
Enzymatic formation of α
-
keto acids.....................
......................................
.............................................
.
.......................................
...
...
...
2
Table S5
.
Isosbestic point of tryptophan analogs and the corresponding indole analogs.
......................................................................
.............................
..........
...
....2
Table S
6
.
Tryptophan analogs synthesized in pre
parative reactions
.
................................................................................................
...
............................................
...
.3
Table S7
.
Optimization of reaction yields.
..............................................
....................
..............................
............................................................................................
..
4
Figure S1
.
Locations of mutations in
Pf
TrpB
7E
6
........................................................
........
......................
.............................................................................................
..
5
Figure S2.
Characterization of
Pf
TrpB
7E6
.
............................................................................
....................
..........
..................................................................................
..
5
Figure S3.
Substrate binding and conformational changes in
Pf
TrpB
with iPrSer.
............................................
.............................
.........
.........................................
...
.6
Experimental Procedures
.............................................................................................................................
......
.......
................................................
........................
..
6
Table S8
.
Primers for random mutagenesis.
.............................................................................................................................
.......
.........
.........................................
...
7
Table S9
.
Summary of the
mutations
that were subjected to recombination
and their variant
-
of
-
origin................................................................................
....
..........
..
7
Table S10
.
Primers for cloning recombination libraries.
....
...................
..............................
................
..
..............................................................................................
...
7
Table S11
.
Primers for site
-
directed and site
-
saturation mutagenesis.
.....................
.............................................................................................
...............................7
Table
S
12
.
Crystallographic data collection and refinement statistics.
............................................................
......................................................
..............................13
Product Characterization
.............................................................................................................................
....................
....................................
..............................14
Results and Discussion
Table S1
.
Summary of the mutations (green) included in the recombination library 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.
The T
50
values and
standard
errors were determined by fitting the data from two independent replicates.
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
.
Engineering
Pf
TrpB
through directed evolution for improved β
-
EtTrp production. Engineering began with
Pf
TrpB
2B9
(
Pf
TrpB
I16V, E17G, I68V, F95L,
F274S, T292S, T321A, and V384A) with 80 TTN. All reactions were performed in at least duplicate with 0.1% catalyst loading fo
r 24 h
ours at 75 °C.
Variant
Mutations
Added
Mutations
Removed
Average
TTN
[a]
Pf
TrpB
2B9
L161A
L161A
N/A
800
±
10
[b]
Pf
TrpB
0E3
L91P
N/A
3400
± 70
[b]
Pf
TrpB
8C8
V173E
N/A
4300
±
300
[c]
Pf
TrpB
7E6
F274L
I68V, T321A
4600
±
200
[c]
Pf
TrpB
2G8
M139L, N166D, S335N
L
91P
3800
±
300
[a] Site
-
directed mutagenesis. [b] Random mutagenesis. [c] Recombination.
SUPPORTING INFORMATION
2
Table
S4
.
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 unde
r 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
Table
S5
.
Isosbestic point of tryptophan analogs and the corresponding indole analog
s
.
Nucleophi
le 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
SUPPORTING INFORMATION
3
Table S6
.
Tr
yptophan analogs synthesized in preparative reactions. Preparative reactions were performed with 100 μmol of nucleophile and
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
SUPPORTING INFORMATION
4
Table
S7
.
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
(0.01%
–
0.1%). Reactions were incubated for 24
h
at 75
°
C and analyzed by LCMS.
Enzyme
Catalyst Loadi
ng (%)
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
-
E
tTrp
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
SUPPORTING INFORMATION
5
Figure S1.
Locations of mutations in
Pf
TrpB
7E
6
.
Pf
TrpB
7E6
(PDB: 6CUV)
contains nine mutations relative to wild
-
type
Pf
TrpB. Mutat
ions 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
T
TN 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 duplicate.
SUPPORTING INFORMATION
6
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 s
hown
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 car
bon 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) relative to tetramethylsilane and calibrated using the residu
al solvent
resonance (DMSO, δ 2.50 ppm). D
ata are reported as follows: chemical shift (multiplicity [singlet (s), doublet (d), doublet of doublets
(dd), doublet of doublets of doublets (ddd), triplet (t), triplet of doubles (td), multiplet (m)], coupling constants [Hz], i
ntegration).
Carbon NMR sp
ectra were recorded with complete proton decoupling. Carbon chemical shifts are reported in ppm relative to
tetramethylsilane and calibrated using the residual solvent proton resonance as an absolute reference. All NMR spectra were
recorded at ambient temp
erature (about 25 °C). Preparative reversed
-
phase chromatography was performed on a Biotage Isolera
One purification system, using C
-
18 silica as the stationary phase, with CH
3
OH as the strong solvent and H
2
O (0.1% HCl by weight)
as the weak solvent. Liqui
d 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, T292S, F274S, T321A, F95L, I16V, V384A)
was cloned and expressed as described previously
.
[2]
Subst
rate 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 rota
mer
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 mutagen
esis 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
.
[3]
PCR fragments were
treated with DpnI for 2 h at 37
°
C, purified by gel extracti
on, and then inserted into a pET
-
22b(+) vector via Gibson assembly
.
[4]
BL21(DE3) E. cloni
®
Express cells were transformed with the Gibson assembly product.
SUPPORTING INFORMATION
7
Table S8
.
Pri
mers for random mutagenesis.
Primer
Sequence (5’ to 3’)
Random mutagenesis forward (NdeI)
GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG
Random mutagenesis reverse (XhoI)
GCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAG
pET22
-
b(+) Forward
CATATGTATATCTCCTTCTTAAAGTTAAACAA
AATTATTTC
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, I
68V, 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). Fragmen
ts were treated with DpnI for one hour at 37 °C and purified
by a preparative agarose gel. The individual fragments were used as 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 in
serted into pET
-
22b(+) via Gibson assembly.
[4]
BL21(DE3) E. cloni
®
Express cells were transformed with the library.
Table
S
9
.
Summary of the
mutations
that were subjecte
d 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
10
.
Primers for cloning recombination libraries.
Fragmen
t
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
GGCTCTGCGTGATTGGGWAGCTAC
TTTTGAATA
TTCTTCATCGGTTACTGYCACGTATTCAGCAC
T321 to XhoI
GTGCTGAATACGTGRCAGTAACCGATGAAGAA
GCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAG
NdeI to I68
GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG
CACGTTTCAGGTATAYTTTAGCACCACCG
I68 to F274
CGGTGGTGCTAAARTATACCTGAAACGTG
GA
CAGCATGCCATGMRACACACCAACCTGACC
F274 to V384
GGTCAGGTTGGTGTGTYKCATGGCATGCTGTC
GAGCACGTTGCCAGATRCTTTCAGGACAATATC
V384 to XhoI
GATATTGTCCTGAAAGYATCTGGCAACGTGCTC
GCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAG
Site
-
directed and site
-
saturation mutagenesis.
Site
-
direct
ed 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 descr
ibed previously.
[5]
Constructs were used to
transform BL21(DE3) E. cloni
®
E
xpress cells.
Table S11
.
Primers for site
-
directed and 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
-
s
aturation
CGTAATTCCAGTTAACTCCGGTTCTCGCACC
XXX
AAAGACGCAATCAACG
GGTGCGAGAACCGGAGTTAACTGGAATTACGTTT
GC
Pf
TrpB
7E6
A161V
TTCTCGCACCGTGAAAGACGCAA
CCGGAGTTAACTGGAATTACGTTTG
XXX in site saturation primers denotes NDT, VHG, or TGG.
[5]
Variant DNA Sequencing.
Variants identified through screening were DNA sequenced to determine their ident
ities. 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
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAATTCAA
TCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACTGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTG
GGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGCGAAAGACGCAATCAACGAGGCT
SUPPORTING INFORMATION
8
CTGCGTGATTGGGTGGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGG
CTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTCCCATGGCATGCTGTCCTACT
TTCTGCAGGACGAAGAAGGTCAGATCAAACCAAG
CCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGGCTGTAACCGATGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTAC
CGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGAT
CATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGAC
CTGGATATTGTCCTGAAAGCGTCTGGCAACGTGCTCGAGCACCACCA
CCACCACCACTGA
Pf
TrpB
2B9
L161G
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAATTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGAC
TGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACTGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAG
CTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGGGAAAGACGCAATCAACGAGGCT
CTGCGTGATTGGGTGGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTA
ACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTCCCATGGCATGCTGTCCTACT
TTCTGCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCG
TGCTGAATACGTGGCTGTAACCGATGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTAC
CGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGAT
CATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGTCCTGAAAGCGTCTGGCAACGTGCTCGAGCACCACCA
CCACCACCACTGA
Pf
TrpB
2B9
L161V
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAATTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCA
ACA
ACGCCATCGGTCAGGCACTGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGTGAAAGACGCAATCAACGAGGCT
CT
GCGTGATTGGGTGGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGT
AAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTCCCATGGCATGCTGTCCTACT
TTCTGCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGGCTGTAACCGATGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTAC
CGAAGGTA
TCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGAT
CATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGTCCTGAAAGCGTCTGGCAACGTGCTCGAGCACCACCA
CCACCACCACTGA
Pf
TrpB
0E3
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAA
AGCTTACAAACGT
TTCAAAGATGACGAAGAATTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACCGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAG
CACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGCGAAAGACGCAATCAACGAGGCT
CTGCGTGATTGGGTGGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCA
TCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTCCCATGGCATGCTGTCCTA
CT
TTCTGCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGGCTGTAACCGACGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTA
CCGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGA
TC
ATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGTCCTGAAAGCGTCTGGCAACGTGCTCGAGCACCACC
ACCACCACCACTGA
Pf
TrpB
8C8
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTTGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAGTTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCC
AACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAGTCTACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACCGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAA
CGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGCGAAAGACGCAATCAACGAGGCT
CTGCGTGATTGGGAGGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATG
TAATCGTTGCTT
SUPPORTING INFORMATION
9
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTCCCATGGCATGCTGTCCTACT
TTCTGCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCC
AGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGGCTGTAACCGACGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTA
CCGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGA
TCATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGTCCTGAAAGCGTCTGGCAACGTGCTCGAGCAC
CACC
ACCACCACCACTGA
Pf
TrpB
7E6
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTAGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAGTTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAATATACCTGAAACGTGAAGACCTGG
TTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACCGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGC
GAAAGACGCAATCAACGAGGCT
CTGCGTGATTGGGAAGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAG
CTGGTTGGCGTTGAGGCTG
GTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTTGCATGGCATGCTGTCCTACT
TTCTGCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGACAGTAACCGATGAAGAAGCACTGAAAGCGTTCC
ATGAACTGAGCCGTAC
CGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGAT
CATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGTCCTGAAAGCATCTGGCAACGTGCTCGAGCACCACCA
CCACCACCACTGA
Pf
TrpB
7E6
L161V
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAAC
GCTGGTAGGACCCCTGAAAGAGCTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAGTTCAATCGTCAGCTGAATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAATATACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACCGCTGGCAAAGCTCATGGGTAAAACT
CGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGTGGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAAATGAAC
GTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGTGAAAGACGCAATCAACGAGGCT
CTGCGTGATTGGGAAGCTACTTTTGAATACACCCACTACCTAATCGGTT
CCGTGGTCGGTCCACATCCGTATCCGACCATCGTTC
GTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAGATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCTT
GTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCTG
GTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCA
GGTTGGTGTGTTGCATGGCATGCTGTCCTACT
TTCTGCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCGCACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACACG
CTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGACAGTAACCGATGAAGAAGCACTGAAAGCGTTCCATGAACTGAGCCGTAC
CGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAA
CTGGCTAAGGAAATGTCTCGTGATGAGAT
CATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGTCCTGAAAGCATCTGGCAACGTGCTCGAGCACCACCA
CCACCACCACTGA
Pf
TrpB
2G8
ATGTGGTTCGGTGAATTTGGTGGTCAGTACGTGCCAGAAACGCTGGTAGGACCCCTGAAAGAGTTGGAAAAAGCTTACAAACGT
TTCAAAGATGACGAAGAGTTCAATCGTCAGCTGA
ATTACTACCTGAAAACCTGGGCAGGTCGTCCAACCCCACTGTACTACGCAA
AACGCCTGACTGAAAAAATCGGTGGTGCTAAAATATACCTGAAACGTGAAGACCTGGTTCACGGTGGTGCACACAAGACCAACA
ACGCCATCGGTCAGGCACTGCTGGCAAAGCTCATGGGTAAAACTCGTCTGATCGCTGAGACCGGTGCTGGTCAGCACGGCGTA
GCGACTGCAATGGCTGGTGCACTGCTGGGCATGAAAGT
GGACATTTACATGGGTGCTGAGGACGTAGAACGTCAGAAACTGAA
CGTATTCCGTATGAAGCTGCTGGGTGCAAACGTAATTCCAGTTAACTCCGGTTCTCGCACCGCGAAAGACGCAATCGACGAGGC
TCTGCGTGATTGGGAAGCTACTTTTGAATACACCCACTACCTAATCGGTTCCGTGGTCGGTCCACATCCGTATCCGACCATCGTT
CGTGATTTTCAGTCTGTTATCGGTCGTGAGGCTAAAGCGCAG
ATCCTGGAGGCTGAAGGTCAGCTGCCAGATGTAATCGTTGCT
TGTGTTGGTGGTGGCTCTAACGCGATGGGTATCTTTTACCCGTTCGTGAACGACAAAAAAGTTAAGCTGGTTGGCGTTGAGGCT
GGTGGTAAAGGCCTGGAATCTGGTAAGCATTCCGCTAGCCTGAACGCAGGTCAGGTTGGTGTGTTGCATGGCATGCTGTCCTAC
TTTCTGCAGGACGAAGAAGGTCAGATCAAACCAAGCCACTCCATCG
CACCAGGTCTGGATTATCCAGGTGTTGGTCCAGAACAC
GCTTACCTGAAAAAAATTCAGCGTGCTGAATACGTGACAGTAACCGATGAAGAAGCACTGAAAGCGTTCCATGAACTGAACCGTA
CCGAAGGTATCATCCCAGCTCTGGAATCTGCGCATGCTGTGGCTTACGCTATGAAACTGGCTAAGGAAATGTCTCGTGATGAGA
TCATCATCGTAAACCTGTCTGGTCGTGGTGACAAAGACCTGGATATTGT
CCTGAAAGCATCTGGCAACGTGCTCGAGCACCACC
ACCACCACCACTGA
Protein expression and purification.
A single colony containing the appropriate
Pf
TrpB gene was used to inoculate 5 mL
Terrific Broth supplemented with 100 μg/mL ampicillin (TB
amp
) and incubated overnight at 37 °C and 230 rpm. For expression, 2.5 mL
of overnight culture were used to inoculate 250 mL TB
amp
in a 1
-
L flask and incubated at
37 °C and 250 rpm for 3 h to reach OD
600
0.6 to 0.8. Cultures were chilled on ice for 20 min and expression was induced with a final concentration of 1 mM isopropyl β
-
D
-
thiogalactopyranoside (IPTG). Expression proceeded at 25 °C and 250 rpm for approximat
ely 20 h. Cells were harvested by
centrifugation at 5,000
g
for 5 min at 4 °C, and then the supernatant was decanted. The pellet was stored at −20 °C until further use.
SUPPORTING INFORMATION
10
Thawed cell pellets were resuspended in 9 mL of lysis buffer containing 25 mM potassium
phosphate buffer, pH 8.0 (KPi buffer)
with 100 mM NaCl, 20 mM imidazole, 1 mg/mL hen egg white lysozyme (HEWL), 200 μM pyridoxal phosphate (PLP), 2 mM MgCl
2
,
0.02 mg/mL DNase I. Pellets were completely resuspended and then lysed with 1 mL BugBuster
®
accord
ing to manufacturer’s
recommendations. Lysate was heat treated at 75 °C for 15 min. The supernatant was collected from clarified lysate following
centrifugation for 15 min at 15,000
g
and 4 °C. Purification was performed with an AKTA purifier FPLC system (G
E Healthcare) and a
1
-
mL Ni
-
NTA column. Protein was eluted by applying a linear gradient of 100 mM to 500 mM imidazole in 25 mM KPi buffer, pH 8.0
and 100 mM NaCl. Fractions containing purified protein were dialyzed into 50 mM KPi buffer, pH 8.0, flash fro
zen in liquid nitrogen,
and stored at −80 °C. Protein concentrations were determined using the Bio
-
Rad Quick Start™ Bradford Protein Assay.
Library expression and screening.
Single colonies from libraries containing the appropriate
Pf
TrpB variant genes wer
e
expressed in 96
-
well deep
-
well plates containing 300 μL of TB
amp
and incubated overnight (approximately 20 h) at
37
°
C and 250 rpm
with 80% humidity. For expression, 20 μL of overnight culture were transferred into 630 μL TB
amp
and incubated for 3 h at 3
7
°
C and
250 rpm with 80% humidity. Cells were then chilled on ice for 20 min and induced with 50 μL of IPTG in TB
amp
(0.5 mM
–
1 mM final
concentration), followed by overnight incubation at
25
°
C and 250 rpm. Cells were harvested by centrifugation at 4
°
C a
nd 4,000
g
for
15 min and then stored at −20
°
C for at least 24 h. Cell plates were thawed and resuspended in 400 μL/well 50 mM KPi buffer, pH 8.0
with 1 mg/mL HEWL, 100 μM PLP, 2 mM MgCl
2
, and 0.02 mg/mL DNase. Cells were lysed by a 30
–
60
-
min incubation at
37 °C and
heat treatment in a 75 °C water bath for 20 min. Lysate was clarified by centrifugation at 5,000
g
for 10 min.
Reactions were performed in a UV
-
transparent 96
-
well assay plate with a total volume of 200 μL/well comprised of 20
–
40 μL
heat
-
treated
lysate, 500 μM indole, and 5 mM β
-
DL
-
ethylserine
(
Sigma)
in 50 mM KPi buffer, pH 8.0. Due to the racemic nature of the
substrate, the effective concentration of β
-
L
-
ethylserine is 2.5 mM. Reactions proceeded in a 75 °C water bath and were assessed for
prod
uct formation at multiple time points (0.5
-
4 h). Prior to being measured, plates were cooled on ice and centrifuged briefly to
collect condensation and assayed by measuring absorption at 290 nm.
UV
-
Vis Spectroscopy.
Spectra were collected on a Shimadzu UV1
800 spectrophotometer in a quartz cuvette with a 1 cm path
length at 75 °C.
Steady
-
state distribution of catalytic intermediates.
Spectra were collected between 250 nm and 500 nm immediately following
substrate addition. Samples were prepared in a total v
olume of 400 μL with 20 μM purified enzyme and 20 mM substrate (threonine,
β
-
L
-
ethylserine, β
-
L
-
propylserine
(Enamine)
) in 50
–
200 mM KPi buffer, pH 8.0. Data were baseline subtracted and normalized to the
E(Ain) peak at 412 nm. Catalytic intermediates were
assigned at the following wavelengths: E(Ain) at 412 nm, E(Aex
1
) at 428 nm,
and E(A
-
A) at 350 nm.
Deamination of the amino
-
acrylate.
Spectra were collected between 250
–
550 nm immediately following substrate addition, and
then once per min for ten min. Sam
ples were prepared in a total volume
of 400 μL with 20 μM purified enzyme and 20 mM substrate
(Threonine, β
-
L
-
ethylserine, β
-
L
-
propylserine) in 50
–
200 mM KPi buffer, pH 8.0. Data were baseline subtracted and plotted as
absorbance over time where α
-
keto acid formation is represented by the slo
pe. Deamination is described in AU/min as the extinction
coefficient is unknown for β
-
L
-
ethylserine and β
-
L
-
propylserine.
Isosbestic points.
Spectra were collected between 250 nm and 550 nm immediately following
Ser
addition, and then once per
min for ten
min. Samples were prepared in a total volume of 400 μL with 1 μM of purified enzyme and 100 μM
–
1 mM nucleophile
SUPPORTING INFORMATION
11
substrate in 50 mM KPi buffer, pH 8.0. The isosbestic point was defined as the overlapped position of the starting material a
nd
product UV peaks
. The isosbestic point of some nucleophiles have been reported previously.
[2]
Small
-
scale analytical reactions.
All analytical reactions were performed in 2
-
mL glass HPLC vials charged with
nucleophile substrate, followed by addition of amino acid s
ubstrate and purified enzyme in 50 mM KPi buffer, pH 8.0 to a final volume
of 150 μL. Reactions were incubated in a 75 °C water bath for 24 h. The reaction was then diluted with 850 μL of 1:1 1
-
M aq.
HCl/CH
3
CN and vortexed thoroughly. The reaction mixture
was then subjected to centrifugation at >20,000
g
for 10 min and the
supernatant analyzed by HPLC. Yields were determined at the relevant isosbestic point
(
Table S5
)
and calculated as area of the
product peak divided by the sum of the integrated product and
substrate peaks. All reactions were performed at least in duplicate.
TTN determination.
A 2
-
mL glass HPLC vial was charged with 20 mM nucleophile substrate as 6 μL of a 500
-
mM solution in
DMSO. Next, 20 mM amino acid substrate and
then
2 μM purified enzym
e (0.01% catalyst loading, 10,000 max TTN) were added as
solution
s
in 50 mM KPi buffer, pH 8.0. The reactions were worked up and analyzed as described above. TTN were determined as
yield times max TTN.
Coupling efficiency.
A 2
-
mL glass HPLC vial was charge
d with 20 mM nucleophile substrate as 6 μL of a 500
-
mM solution
in DMSO. Next, 20 mM amino acid substrate and 20 μM purified enzyme (0.1% catalyst loading, 1,000 max TTN) were added as a
solution in 50 mM KPi buffer, pH 8.0. Coupling efficiency was describ
ed as the yield under reaction conditions with high catalyst
loading and equimolar substrate equivalents.
Synthesis and characterization of tryptophan analogs.
Preparative reactions were carried out by adding 100 μmol of
nucleophile substrate and 200 μmo
l
L
-
amino acid substrate to a 40
-
mL reaction vial. Following substrate addition, 10 mL of 50 mM
KPi buffer, pH 8.0 containing purified
Pf
TrpB
7E6
or
Pf
TrpB
2G8
(
0.01
–
0.4% catalyst loading
) were added to the reaction vial
. The
reaction mixture was incubated i
n a 75 °C water bath for 24 h, frozen on dry ice, and then the water was removed by lyophilization.
Approximately 4 mL of 1:1 CH
3
CN/1 M aq. HCl were added to the remaining solid and the volume was reduced
in vacuo
. The sample
was resuspended in water and l
oaded onto a 12g C
-
18 column equilibrated with 1% methanol/water (0.1% HCl by mass) on a
Biotage Isolera One purification system. The column was washed with three column volumes (CV) of 1% methanol/water mixture.
The product was the eluted with a gradient
from 1% to 100% methanol over 10 CV. The fractions containing the UV
-
active product
were combined and the volume reduced
in vacuo
. The product was then suspended in water (0.1% HCl by mass) and transferred to
a tared vial before being frozen on dry ice and
lyophilized. Yields were determined by product mass following lyophilization relative to
theoretical yield with indole analog as the limiting reagent. Products were obtained as hydrochloride salts and product ident
ities were
confirmed by
1
H
-
and
13
C
-
NMR a
nd high
-
resolution mass spectrometry.
Determination of Optical Purity.
Product optical purity was estimated by derivatization with FDNP
-
alanamide. Approximately
0.5 μmol of purified β
-
MeTrp, β
-
Et
Trp
, or β
-
Pr
Trp were added to a 2
-
mL vial. The product was re
suspended in 100 μL of 1 M aq.
NaHCO
3
. FDNP
-
alanamide (10 μL of a 33
-
mM solution in acetone, 0.33 μmol) was added to each vial, followed by a two
-
hour
incubation at 37 °C and 230 rpm. The reaction mixture was then cooled to room temperature and diluted wit
h 1:1 CH
3
CN/1
-
M aq. HCl
(600 μL). The resulting solution was analyzed directly by LCMS at 330 nm. Each amino acid was derivatized with both racemic a
nd
enantiopure FDNP
-
alanamide for comparison. Absolute stereochemistry was inferred by analogy to
L
-
tryptop
han. All products were
>99% ee.
SUPPORTING INFORMATION
12
Determination of T
50
values.
A solution of 1 μM purified enzyme in 50 mM KPi buffer, pH 8.0 was aliquoted into 12 PCR tubes
with a volume of 95 μL/tube. Ten of these samples were incubated in a thermocycler for 60 min with a
temperature gradient from
75 °C to 95 °C, while the two remaining samples were incubated at room temperature as controls. All 12 tubes were centrifuged
for
three min to pellet precipitated enzyme, and then 75 μL of the supernatant were transferred from ea
ch tube to a UV
-
transparent 96
-
well assay plate. Enzyme activity was determined by adding an additional 75 μL of 50 mM KPi buffer, pH 8.0 containing 1 mM in
dole
and 1 mM serine to each well. Reactions were incubated for 10 min at 75 °C and then briefly cen
trifuged to collect condensation.
Activity was determined by measuring product formation at 290 nm. Activity was correlated to incubation temperature, and
t
hermostability is reported as the temperature at which half of the activity is lost (T
50
) after 1
-
ho
ur incubation.
Measurements were
conducted in duplicate
and data were fitted to determine the T
50
value and error
.
Crystallography.
Seed stocks of wild
-
type
Pf
TrpB
crystals
were used to seed crystallization of
Pf
TrpB
7E6
.
The wild
-
type
Pf
TrpB
crystal was ob
tained
by sitting drop vapor diffusion
against a 1
-
mL reservoir containing 24% PEG3350 and 50 mM Na HEPES, pH
7.85. The seed stock was prepared according to the Seed Bead method (Hampton Research) using 24% PEG3350 and 50 mM Na
HEPES, pH 7.85 as stabilizat
ion buffer. The seed stock was diluted 2,000x in stabilization buffer before use.
Pf
TrpB
7E6
crystals were
grown in sitting drops against a 1
-
mL reservoir of 14% PEG3350 and
0.1 M Na HEPES (pH 7.85) with mother liquor comprised of 1.5
μL of 18.8 mg/mL
Pf
Trp
B
7E6
and 1.5 μL of 2,000x diluted seed stock.
Ligand
-
bound structures were determined by soaking
Pf
TrpB
7E6
crystals with the
compound
of interest. From a 50/50% (v/v) mixture
containing 0.5 M
β
-
DL
-
ethylserine
in 0.2 M KPi buffer, pH 8.0 and stabilization
buffer, 0.5 μL were added to the sitting drop and
incubated for 2 h.
(2
S
)
-
β
-
isopropylserine was soaked into
Pf
TrpB
7E6
crystals by adding powdered substrate directly to the sitting drop,
mixing gently, and incubating for one hour.
Crystals were cryoprotect
ed through oil immersion in Fomblin Y (Sigma) and flash
-
frozen in liquid nitrogen until diffraction.
Diffraction data were collected remotely at the Stanford Synchrotron Radiation Laboratories on beamline 12
-
2.
Crystals routinely
diffracted at or below 2.
2
5
Å, and the data were integrated and scaled using XDS
[6]
and AIMLESS.
[7]
A resolution cutoff of CC
1/2
> 0.3
was applied along the strongest axis of diffraction.
[7,8]
Th
ese data were later judged to
contribute to model quality
based on
R
free
in the
fin
al bin <0.4. Structures were solved using molecular replacement with PHASER, as implemented in CCP4.
[9,10]
The search model
comprised a single monomer of
Pf
TrpB
2B9
(
holo
and
(2
S
, 3
R
)
-
β
-
E
tS
er, PDB: 5VM5) or
Pf
TrpB
4D11
(
(2
S
, 3
S
)
-
β
-
iPrSer)
and was
prepared by removing all ligand, trimmined mutated residues to Cβ, and then
subjected to ten cycles of geometric idealization in
REFMAC5.
[10]
Model
-
building was performed in Coot
[11]
beginning with data processed at 2.4 Å, followed by subsequent inclusion of
increasingly higher
-
resolution shells of data with relaxed geometri
c constraints. This procedure was particularly important for the
structures of β
-
L
-
ethylserine and β
-
L
-
isopropylserine
-
bound
Pf
TrpB
7E6
, which contained a large rigid body motion of the COMM
domain
relative to the search model
. Refinement was performed usin
g REFMAC5
9
.
The MolProbity server was used to identify
rotamer flips and to identify clashes.
[12]
After the protein, ligand, and solvent atoms were built, TLS operators were added to
refinement, which resulted in substantial improvements in R
free
for the models.
Despite these improvements, R
free
were
still high
compared to structures of
similar
resolution. This has proved to be a general feature of TrpB crystals in the spacegroup, and we
hypothesize the high R factors are caused by the considerable domain
-
level conformational heterogeneity that is diff
icult
to
parameterize with a single structure.
[13]
This heterogeneity
was
increased when ligands
were
soaked into
Pf
TrpB
7E6
and
bound
with
incomplete occupancy
. We observed much weaker density in the C
OMM domain of several protamers, which led to an uncommonly