nonribosomal biosynthesis of backbone-modified peptides · nonribosomal biosynthesis of...
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Nonribosomal biosynthesis of backbone-modified peptides
David L. Niquille, Douglas A. Hansen, Takahiro Mori, David Fercher, Hajo Kries, Donald Hilvert*
Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland
*e-mail: [email protected]
Materials and Methods 3
Chemistry 3
Biocatalysis 8
Small Molecule X-ray Crystallography 9
Biology 10
Cloning 10
Yeast Surface Display 10
Library Construction 11
4’-Phosphopantetheinyl Transferase Sfp 11
TycAβpY and TycAβF 11
TycAβpY-AN and TycAβF-AN 12
TycB1-SrfTEP26G 12
GrsB 13
High-Throughput Adenylation and Thioesterification Assay 14
Protein Production 16
Protein Purification 16
Identification of a Second Start Site in grsB 17
Pyrophosphate Exchange Assay 18
Crystallization and Structure Determination of β-A Domains 18
Dipeptide Synthetase Reactions 18
Pentapeptide Synthetase Reactions 19
In Vivo Pentapeptide Production 20
Supplementary Figures 21
Supplementary Figure 1. A high-throughput assay for adenylation and thioesterification 21
Supplementary Figure 2. Sequences of selected O-propargyl-(S)-β-Tyr-specific TycA variants 22
Supplementary Figure 3. TycA purification and characterization 23
Supplementary Figure 4. Active sites of TycAβpY-AN and TycAβF-AN 24
Supplementary Figure 5. Structural rationale for the α/β-switch 25
1© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2891
NATURE CHEMISTRY | www.nature.com/naturechemistry 1
Supplementary Figure 6. β-Amino acid binding mode 26
Supplementary Figure 7. In vitro formation of pentapeptide 11 and gramicidin S 27
Supplementary Figure 8. In vivo formation of pentapeptide 11 28
Supplementary Figure 9. Active site sequence alignments of representative A domains 29
Supplementary Tables 30 Supplementary Table 1. Adenylation steady-state parameters. 30
Supplementary Table 2. Data collection, phasing, and refinement statistics. 31
Supplementary Table 3. Crystal data and structure refinement for compound 9. 32
Supplementary Table 4. Crystal data and structure refinement for compound 10. 33
Supplementary Table 5. DNA sequences encoding the proteins used in this study. 34
NMR Spectra 42
Supplementary References 51
2
Materials and Methods
Chemistry
All reagents were used as received, all solvents were technical grade, and all reactions were run in open
flasks fitted with PFTE coated magnetic stir bars at room temperature (RT) unless otherwise noted.
Analytical thin-layer chromatography (TLC) was performed with Merck 60 F254 pre-coated glass plates
(0.25 mm) and visualized using a combination of UV detection (254 nm), p-anisaldehyde, and KMnO4
stains. RT reactions were conducted at ~23 °C, reactions run cooler than RT were performed in a cold
room (4 °C), an ice bath (0 °C), or a NaCl/ice bath (-10 °C). Flash column chromatography was
performed using SiliCycle (SilaFlash® P60, 230 – 400 mesh particle size) silica gel. Preparative HPLC
was performed on a Waters system consisting of 515 pumps in line with a 2487 dual λ absorbance
detector and a fraction collector using a Reprosil-Pur 120 C18-AQ column (150 x 20mm, 5 μm, Dr.
Maisch GmbH, Basel, Switzerland). High resolution mass spectrometry (HRMS) was performed on a
Bruker maXis UHR-TOF by electrospray ionization (ESI) or a Bruker solariX by matrix-assisted laser
desorption/ionization (MALDI) at the Mass Spectrometry Service of the Laboratory of Organic
Chemistry (LOC) at ETH Zurich. NMR spectra were recorded on a Bruker Advance-III 400 MHz
spectrometer at the NMR Service at the LOC, ETH Zurich. 1H NMR spectra were recorded relative to
residual solvent peak (CDCl3 δH 7.26 ppm, D2O δH 4.79, D6-DMSO δH 2.50 ppm) and reported as follows:
chemical shift (ppm), multiplicity, coupling constant (Hz), and integration. Multiplicity abbreviations are
as follows: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, h = hextet, ovlp = overlap, br =
broad signal. 13C NMR spectra were recorded relative to residual solvent peaks (CDCl3 δC 77.0 ppm, D6-
DMSO δC 39.5 ppm).
13: 4-hydroxybenzaldehyde (12, TCI, 4.0 g, 32.7 mmol, 1.0 equiv) was dissolved in DMF at RT (Sigma,
33 mL, 1 M) followed by the addition of K2CO3 (Fisher, 5.4 g, 39.2 mmol, 1.2 equiv) in one portion.
Propargyl bromide (80% solution in PhMe, Sigma, 5.8 g, 4.4 mL, 39.2 mmol, 1.2 equiv) was added
dropwise at RT to give a brown solution that was stirred for 24 h. The reaction was quenched with
aqueous NaHCO3 (sat.) and the resulting solution was extracted with EtOAc (3x). The combined organic
extracts were washed with brine and filtered through a sodium sulfate plug, which was subsequently
rinsed with EtOAc (2x), and concentrated. The crude product was dissolved in a minimum amount of
EtOAc and subsequently precipitated with hexanes. The solid was collected via vacuum filtration through
3
a fritted glass funnel, yielding 13 (4.6 g, 28.7 mmol, 88% yield) as a light orange solid that was carried
onto the next step without additional purification. 1H NMR (400 MHz; CDCl3): δ 9.90 (s, 1H), 7.87-7.84
(m, 2H), 7.11-7.07 (m, 2H), 4.78 (d, J = 2.4 Hz, 2H), 2.57 (t, J = 2.4 Hz, 1H). 13C NMR (101 MHz;
CDCl3): δ 190.7, 162.3, 131.9, 130.6, 115.2, 77.5, 76.3, 55.9. MALDI HRMS: calculated [M+H]+
161.0597, found 161.0597.
14: Adapted from literature procedure48, (R)-t-butyl sulfinamide (TCI, 1.8 g, 15 mmol, 1.2 equiv) and 13
(2.0 g, 12.5 mmol, 1.0 equiv) were charged into a flame dried flask under N2 and dissolved in THF
(anhydrous, Sigma, 125 mL, 0.1 M). B(OCH2CF3)3 (7.7 g, 25 mmol, 5.3 mL, 2.0 equiv) was added
dropwise at RT and the reaction was stirred for 2 h before being quenched with NaHCO3 (sat.). The
resulting solution was extracted with EtOAc (3x), the combined organic extracts were washed with brine
and filtered through a sodium sulfate plug, which was subsequently rinsed with EtOAc (2x), and
concentrated. Flash chromatography (EtOAc/hexanes 10:90 to 20:80) afforded 14 (3.1 g, 11.8 mmol,
95%) as a colourless oil. 1H NMR (400 MHz; CDCl3): δ 8.52 (s, 1H), 7.83-7.81 (m, 2H), 7.06-7.04 (m,
2H), 4.76 (d, J = 2.4 Hz, 2H), 2.56 (t, J = 2.4 Hz, 1H), 1.25 (s, 9H).13C NMR (101 MHz; CDCl3):
δ 161.6, 160.8, 131.2, 128.0, 115.2, 77.8, 76.1, 57.6, 55.9, 22.6. ESI HRMS: calculated [M+H]+
264.1053, found 264.1054.
15: Adapted from literature procedure49, zinc (Sigma, powder, 11.0 g, 168.0 mmol, 10.0 equiv) was
charged into a flame dried three-neck flask fitted with a water condenser and a thermometer under N2 and
suspended in anhydrous THF (30 mL). t-Butylbromoacetate (200 μL) was added, followed by DIBAL-H
(1 M solution in PhMe, Sigma, 0.8 mL, 0.8 mmol, 0.05 equiv). The solution was warmed to 40 °C and
additional t-butylbromoacetate (Fluorochem, 8.2 g, 6.4 mL, 42.0 mmol, 2.5 equiv) was added dropwise
while keeping the internal temperature <50 °C. After complete addition, the solution was cooled to -10 °C
4
and 14 (4.4 g, 16.8 mmol, 1.0 equiv) in THF (5 mL, ~0.5 M total) was added dropwise. After complete
addition, the reaction was warmed to 4 °C and stirred for 14 h before being quenched with brine, where
the solution solidified. EtOAc was added and the heterogeneous biphasic mixture was filtered through
celite, followed by EtOAc (3x) rinse. The aqueous layer was separated and the organic layer was washed
with citric acid (sat.). The organic extract was filtered through a sodium sulfate plug, which was
subsequently rinsed with EtOAc (2x), and concentrated. Flash chromatography (EtOAc/hexanes 30:70 to
50:50) afforded 15 (5.2 g, 13.7 mmol, 81%) as a colourless oil. 1H NMR (400 MHz; CDCl3): δ 7.27-7.25
(m, 2H), 6.94-6.92 (m, 2H), 4.71-4.70 (m, 1H), 4.68-4.67 (m, 2H), 4.56 (br d, J = 3.8 Hz, 1H), 2.74-2.72
(m, 2H), 2.51 (t, J = 2.4 Hz, 1H), 1.39 (s, 9H), 1.21 (s, 9H). 13C NMR (101 MHz; CDCl3): δ 170.6, 157.3,
133.9, 128.7, 115.0, 81.8, 78.6, 75.7, 56.0, 55.7, 55.2, 43.8, 28.2, 22.8. ESI HRMS: calculated [M+H]+
380.1890, found 380.1889.
3: A flask was charged with 15 (2.8 g, 7.4 mmol, 1.0 equiv) which was subsequently dissolved in dioxane
(3.7 mL, 0.5 M). Concentrated HCl (12.1 M, Sigma, 18.4 mL, 73.7 mmol, 10.0 equiv) was added slowly
and the resulting solution was stirred for 24 h at RT before being concentrated under a stream of N2. The
crude product was dissolved in a minimum amount of CH2Cl2 and subsequently precipitated with Et2O.
The solid was collected via vacuum filtration through a fritted glass funnel, yielding 3 (1.8 g, 7.0 mmol,
95% yield) as a light yellow solid. 1H NMR (400 MHz; D2O): δ 7.49-7.45 (m, 2H), 7.17-7.14 (m, 2H),
4.84 (d, J = 2.4 Hz, 2H), 3.22-3.06 (m, 2H), 2.97 (t, J = 2.4 Hz, 1H), 1.32 (s, 1H). 13C NMR (101 MHz;
D2O): δ 173.6, 157.5, 128.6, 128.5, 115.7, 78.4, 76.8, 56.0, 51.0, 37.7, 24.3 ESI HRMS: calculated
[M+H]+ 220.0968, found 220.0971.
16: A flask was charged with 3 (1.0 g, 3.9 mmol, 1.0 equiv) and H2O/THF (1:1, 39 mL, 0.1 M), NaOH
(0.3 g, 11.7 mmol, 3.0 equiv) was added in a single portion, and the solution was stirred at RT until
homogenous. Di-t-butyl dicarbonate (Chem Impex, 0.9 g, 4.3 mmol, 1.1 equiv) was added in a single
5
portion and the reaction was stirred for 24 h at which point the solution was concentrated by half under a
stream of N2. The resulting aqueous solution was washed with Et2O:hexanes (1x, 1:1), acidified to
~pH 2-3 with H3PO4 and the resulting solution was extracted with CH2Cl2 (3x). The combined organic
extracts were filtered through a sodium sulfate plug, which was subsequently rinsed with CH2Cl2 (2x),
and concentrated to yield 16 (1.2 g, 3.6 mmol, 92%). 1H NMR (400 MHz; D6-DMSO): δ 12.15 (s, 1H),
7.36 (d, J = 8.7 Hz, 1H), 7.25-7.21 (m, 2H), 6.93-6.89 (m, 2H), 4.85-4.83 (m, 1H), 4.76 (d, J = 2.4 Hz,
2H), 3.53 (t, J = 2.4 Hz, 1H), 2.67-2.51 (m, 2H), 1.34 (br s, 9H). 13C NMR (101 MHz; D6-DMSO): δ
171.8, 156.1, 154.6, 135.9, 127.5, 114.4, 79.3, 78.1, 77.7, 55.3, 50.5, 41.3, 28.2. ESI HRMS: calculated
[M+Na]+ 342.1312, found 342.1311.
6: Adapted from literature procedure50, a flask was charged with 16 (0.32 g, 1.0 mmol, 1.1 equiv), N-
hydroxysuccinimide (Sigma, 0.14 g, 1.2 mmol, 1.32 equiv), EDC•HCl (Chem Impex, 0.22 g, 1.2 mmol,
1.32 equiv), and CH2Cl2 (5 mL, 0.1 M). The reaction was stirred for 4 h before addition of H2O. The
organic layer was separated and the resulting solution was extracted with CH2Cl2 (2x). The combined
organic extracts were filtered through a sodium sulfate plug, which was subsequently rinsed with CH2Cl2
(2x), and concentrated to yield the crude NHS ester of 16. To this flask was added 17 (0.35 g, 0.91 mmol,
1 equiv), Cs2CO3 (Chem Impex, 0.32 g, 1 mmol, 1.1 equiv), and DMF (9 mL, 0.1 M), and the reaction
was stirred for 12 h before being concentrated. Flash chromatography (MeOH/EtOAc 0:100 to 10:90)
afforded partially purified acylated 17, which was subsequently dissolved in TFA/H2O (5:1, 5 mL) for
48 h before being concentrated under a stream of N2. Crude product was dissolved in H2O and purified by
preparative HPLC (H2O:MeCN + 0.1% TFA, 5% to 25% MeCN over 30 min, flowrate of 10 mL/min) to
yield 6 (0.09 g, 0.14 mmol, 15% yield) as a colourless solid. 1H NMR (400 MHz; CD3OD): δ 8.52-8.26
(m, 2H), 7.39-7.33 (m, 2H), 7.04-6.96 (m, 2H), 6.06 (d, J = 4.5 Hz, 1H), 4.74-4.69 (m, 3H), 4.64 (t, J =
6
7.1 Hz, 1H), 4.56 (t, J = 4.8 Hz, 1H), 4.44-4.32 (ovlp m, 3H), 4.26-4.23 (m, 1H), 3.27 (dt, J = 3.2, 1.6 Hz,
1H), 3.15-2.93 (m, 2H), 2.93-2.89 (m, 1H). 13C NMR (101 MHz; MeOD): δ 169.9, 159.8, 150.0, 145.8,
143.6, 129.8, 129.7, 116.7, 116.6, 90.5, 83.3, 79.4, 77.1, 77.0, 75.8, 72.3, 71.4, 56.7, 52.0, 49.9, 40.5,
39.0. ESI HRMS: calculated [M+H]+ 548.1558, found 548.1558.
5: A flask was charged with 18 (0.25 g, 0.96 mmol, 1.1 equiv), N-hydroxysuccinimide (Sigma, 0.13 g,
1.15 mmol, 1.32 equiv), EDC•HCl (Chem Impex, 0.22 g, 1.15 mmol, 1.32 equiv) and CH2Cl2 (10 mL, 0.1
M). The reaction was stirred for 4 h before addition of H2O. The organic layer was separated and the
resulting solution was extracted with CH2Cl2 (2x). The combined organic extracts were filtered through a
sodium sulfate plug, which was subsequently rinsed with CH2Cl2 (2x), and concentrated to yield the crude
NHS ester of 18. To this flask was added 17 (0.31 g, 0.79 mmol, 1 equiv), Cs2CO3 (Chem Impex, 0.28 g,
0.87 mmol, 1.1 equiv), and DMF (8 mL, 0.1 M), and the reaction was stirred for 12 h before being
concentrated. Flash chromatography (MeOH:acetone 0:100 to 2:98) afforded partially purified acylated
17, which was subsequently dissolved in TFA/H2O (5:1, 5 mL) for 48 h before being concentrated under
a stream of N2. Crude product was dissolved in H2O and purified by preparative HPLC (H2O:MeCN +
0.1% TFA, 5% to 25% MeCN over 30 min, flowrate of 10 mL/min) to yield 5 (0.14 g, 0.22 mmol, 29%
yield) as a colourless solid. 1H NMR (400 MHz; CD3OD): δ 8.52-8.25 (m, 2H), 7.47-7.41 (m, 5H), 6.11
(d, J = 4.5 Hz, 1H), 4.78-4.71 (m, 2H), 4.61 (t, J = 4.8 Hz, 1H), 4.50-4.35 (m, 3H), 4.28 (ddd, J = 4.9, 3.7,
2.9 Hz, 1H), 3.20-3.00 (m, 2H). 13C NMR (101 MHz; CD3OD): δ 170.0, 150.2, 146.0, 143.6, 137.1,
130.7, 130.5, 128.3, 120.5, 90.5, 83.3, 75.8, 72.5, 71.4, 59.7, 52.5, 40.6. ESI HRMS: calculated [M+H]+
494.1452, found 494.1447.
7
Biocatalysis
All buffers were prepared using purified H2O (Nanopure system, Barnstead). Buffer components were
used as received from specified commercial suppliers and used without further purification. TLC, flash
chromatography, NMR spectroscopy, and mass spectrometry were performed as described in the
Chemistry section.
9 (0.15 mmol scale): A 250 mL flask was charged with 3 (0.038 g, 0.15 mmol, 1.0 equiv), L-Pro (Sigma,
0.017 g, 0.15 mmol, 1.0 equiv), and ATP (Sigma, 0.41 g, 0.75 mmol, 5.0 equiv). Buffer was added [2x
concentration, 75 mL, Bis-Tris propane (200 mM), NaCl (200 mM), MgCl2 (20 mM), TCEP (2 mM)],
followed by H2O (62 mL), and the pH was adjusted to 9.0 with NaOH. TycAβpY (55 μM stock, 2 μM
final, 5.5 mL, 0.2 mol %) and TycB1-SrfTEP26G (40 μM stock, 2 μM final, 7.5 mL, 0.2 mol %) were
added to give a final volume of 150 mL. This solution was aliquoted into falcon tubes (3x 50 mL), which
were placed in a preheated 37 °C water bath for 20 min before transfer to a 37 °C incubator for 36 h. The
solution was quenched with acetone (2x v/v) and filtered through a silica plug, washed with acetone (3x).
Acetone was evaporated and the resulting aqueous solution was extracted with EtOAc. The combined
organic extracts were filtered through a sodium sulfate plug, which was subsequently rinsed with EtOAc
(2x), and concentrated. Flash chromatography (EtOAc/acetone 100:0 to 90:10) afforded 9 (0.033 g, 0.11
mmol, 73%) as a colourless solid. 1H NMR (400 MHz; CDCl3): δ 7.26-7.22 (m, 2H), 6.99-6.96 (m, 2H),
5.85 (s, 1H), 4.79 (dd, J = 13.0, 2.2 Hz, 1H), 4.69 (d, J = 2.4 Hz, 2H), 4.58 (dd, J = 8.1, 4.3 Hz, 1H),
3.59-3.56 (m, 2H), 3.18 (t, J = 13.6 Hz, 1H), 2.78-2.68 (m, 2H), 2.53 (t, J = 2.4 Hz, 1H), 2.22-2.13 (m,
1H), 1.91-1.84 (m, 2H). 13C NMR (101 MHz; CDCl3): δ 170.0, 169.0, 157.6, 135.0, 127.1, 115.6, 78.3,
75.9, 59.7, 56.3, 55.9, 46.7, 43.8, 28.7, 23.4. ESI HRMS: calculated [M+H]+ 299.1390, found 299.1395.
10 (0.25 mmol scale): A 50 mL falcon tube was charged with (S)-β-Phe (4, Chem Impex, 0.041 g, 0.25
mmol, 1.0 equiv), L-Pro (Sigma, 0.029 g, 0.25 mmol, 1.0 equiv), and ATP (Sigma, 0.69 g, 1.25 mmol,
8
5.0 equiv). Buffer was added [2x concentration, 25 mL, Bis-Tris propane (200 mM), NaCl (200 mM),
MgCl2 (20 mM), TCEP (2 mM)], followed by H2O (21 mL), and the pH was adjusted to 9.0 with NaOH.
TycAβF (61 μM stock, 2 μM final, 1.7 mL, 0.04 mol %) and TycB1-SrfTEP26G (44 μM stock, 2 μM final,
2.3 mL, 0.04 mol %) were added to give a final volume of 50 mL. The tube was placed in a preheated
37 °C water bath for 36 h and extracted with EtOAc (3x, centrifuged at 4000 x g to break emulsion). The
combined organic extracts were filtered through a sodium sulfate plug, which was subsequently rinsed
with EtOAc (2x) and concentrated. Flash chromatography (EtOAc/acetone 100:0 to 80:20) afforded 10
(0.042 g, 0.17 mmol, 68%) as a white solid.
(1.75 mmol scale): A 500 mL flask was charged with (S)-β-Phe (4, Chem Impex, 0.29 g, 1.75 mmol, 1.0
equiv), L-Pro (Sigma, 1.75 g, 1.75 mmol, 1.0 equiv), and ATP (Meiya pharma, 4.8 g, 8.75 mmol, 5.0
equiv). Buffer was added [2x concentration, 175 mL, Bis-Tris propane (200 mM), NaCl (200 mM),
MgCl2 (20 mM), TCEP (2 mM)], followed by H2O (148 mL), and the pH was adjusted to 9.0 with NaOH.
TycAβF (61 μM stock, 2 μM final, 11.5 mL, 0.04 mol %) and TycB1-SrfTEP26G (44 μM stock, 2 μM final,
16 mL, 0.04 mol %) were added, to give a final volume of 350 mL. This solution was aliquoted into
falcon tubes (7x 50 mL), which were placed in a preheated 37 °C water bath for 20 min, before transfer to
a 37 °C incubator for 36 h. The solution was quenched with acetone (2x v/v) and filtered through a silica
plug, washed with acetone (3x). Acetone was evaporated, and the resulting aqueous solution was
saturated with NaCl before extraction with THF (3x). The combined organic extracts were filtered
through a sodium sulfate plug, which was subsequently rinsed with EtOAc (2x), and concentrated. Flash
chromatography (EtOAc/acetone 100:0 to 80:20) afforded 10 (0.251 g, 1.03 mmol, 59%) as a white solid. 1H NMR (400 MHz; CDCl3): δ 7.41-7.31 (m, 5H), 5.81 (s, 1H), 4.84 (ddd, J = 12.9, 3.0, 0.9 Hz, 1H),
4.61 (dd, J = 8.1, 4.2 Hz, 1H), 3.59 (t, J = 6.8 Hz, 2H), 3.21 (t, J = 13.6 Hz, 1H), 2.80-2.73 (m, 2H), 2.24-
2.14 (m, 1H), 1.93-1.85 (m, 2H). 13C NMR (101 MHz; CDCl3): δ 169.9, 168.8, 141.9, 129.3, 128.6,
125.7, 59.6, 56.8, 46.7, 43.7, 28.6, 23.4 ESI HRMS: calculated [M+H]+ 245.1285, found 245.1287.
Small Molecule X-ray Crystallography. Single crystals of 9 and 10 were obtained by addition of
hexanes to concentrated solutions of Et2O. A suitable crystal was selected and mounted on an XtaLAB
Synergy, Dualflex, Pilatus 300K diffractometer. The crystal was kept at 100.0(1) K during data
collection. Using OLEX251, the structure was solved with the ShelXT52 structure solution program using
Intrinsic Phasing and refined with the XL53 refinement package using Least Squares minimization.
Refinement statistics for compounds 9 and 10 are summarized in Supplementary Tables 3 and 4,
respectively.
9
Biology
All expression media and buffers were prepared using purified H2O (Nanopure system, Barnstead). Media
and buffer components, kits, and enzymes were used as received from specified commercial suppliers. All
commercial enzymes were purchased from NEB if not stated otherwise.
Cloning. All oligonucleotide primers were purchased from Microsynth AG (Switzerland) and all PCR
utilized Phusion HF polymerase (NEB) according to the manufacturer’s instructions using the supplied
HF buffer (50 μL total volume, 50 ng plasmid DNA, 0.5 μM primer, 0.2 mM dNTPs (Sigma), 1 μL
Phusion HF). DNA was either purified via agarose gels (1 %) and extracted using a Zymoclean Gel DNA
Recovery Kit (Zymo Research) or via spin columns using a DNA clean and concentrator kit (Zymo
Research). E. coli transformations were conducted using electrocompetent E. coli HM007937 cells
(100 μL, ~100 ng DNA), followed immediately by SOC (1 mL) rescue and incubation at 37 °C, 400 RPM
for 1 h before plating onto LB agar containing the respective antibiotic. Single colonies were selected and
grown in overnight cultures using LB Miller broth (Merck) containing an appropriate antibiotic. After
harvesting cells by centrifugation, plasmid DNA was isolated with a ZR Plasmid Miniprep Classic kit
(Zymo Research) according to manufacturer specifications. All cloned variants were verified by Sanger
sequencing at Microsynth AG (Switzerland).
Yeast transformations for single variants were conducted with EBY10054 cells using a Frozen-EZ
Yeast Transformation II Kit (Zymo Research) according to manufacturer specifications. To achieve
higher transformation efficiency for the yeast library, EBY100 cells were transformed with linear DNA
following the protocol of Benatuil et al.55. Plasmid DNA from individual yeast colonies was isolated
using a Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research) according to manufacturer
specifications. The DNA segment of interest was PCR amplified from the extracted DNA sample and
confirmed via Sanger sequencing by Microsynth AG (Switzerland).
Yeast Surface Display. Plasmid pCT54 was used for all yeast surface display experiments. To simplify
cloning, the NheI restriction site preceding the sequence encoding the protein of interest was changed to a
NdeI restriction site and the XhoI restriction site of pCT was deleted and reinstalled before the sequence
encoding the c-myc tag. The tycA-AT genes (wild type and W239S) were amplified as two fragments
from plasmids pSU18_tycA and pSU18_tycA_W239S11 using primer pairs tycA-AT_NdeI_f / tycA-
AT_NdeI_del_r and tycA-AT_NdeI_del_f / tycA-AT_XhoI_r to delete an internal NdeI restriction site.
The two fragments were gel purified, assembled by overlap PCR with primer pair tycA-AT2_NdeI_f /
tycA-AT2_NdeI_r, and spin column purified. Introduction into the digested pCT vector via NdeI and
XhoI restriction sites yielded plasmids pCT_tycA-AT and pCT_tycA-AT_W239S.
10
tycA-AT_NdeI_f A TTA CAT ATG GTA GCA AAT CAG G
tycA-AT_NdeI_del_r CGA CTT GCC CTG TGC ATA CGG AAC TAG CGC ATG CTG C
tycA-AT_NdeI_del_f TAT GCA CAG GGC AAG TCG
tycA-AT_XhoI_r C TGA CTC GAG CGT GCT CTT GAC AAA AAG AGC
Library Construction. The TycA library was constructed based on plasmid pCT_tycA-AT_W239S, that
was digested with NdeI and XhoI restriction endonucleases and gel purified. The library was PCR
amplified as three fragments from pCT_tycA-AT_W239S using primer pairs pCT_f / tycA_A236X_r,
tyca_A236X_f / tycA_b13b14_r, and tycA_b13b14_f / pCT_r, which were individually gel purified and
assembled by overlap PCR with primer pair pCT_f / pCT_r. The gel-purified assembly product and
digested pCT vector were directly transformed into electrocompetent EBY10054 cells as described by
Benatuil et al.55, exploiting 100 bp overlaps of vector and insert for homologous recombination.
pCT_f GC TCG ACG ATT GAA GGT AGA TAC C
tycA_A236X_r GTC GAA CGA CAT GCT GGC AAA AAG C
tycA_A236X_f G CTT TTT GCC AGC ATG TCG TTC GAC NNK TCC GTT AGC GAA ATG TTC
ATG GCT TTG C
tycA_b13b14_r TTC CGT CGG GCC GTA TGC ATT TAT GTA CC
tycA_b13b14_f GG TAC ATA AAT GCA TAC GGC CCG ACG GAA NNK NNK NNK GCG ACG ATC
TGG GAA GCC CCG TCC
pCT_r GC TAA AAG TAC AGT GGG AAC AAA GTC G
4’-Phosphopantetheinyl Transferase Sfp. For the production of C-terminally His6-tagged Sfp synthase,
the sfp gene was cloned from genomic DNA of Bacillus subtilis (ATCC 21332) into the pMG21156 vector
using NdeI and XhoI restriction sites. B. subtilis genomic DNA was isolated and purified by isopropanol
precipitation as previously described57 and used to PCR amplify the sfp gene with primer pair sfp_NdeI_f
/ sfp_XhoI_r. The PCR product was spin column purified, digested, and gel purified. Ligation into the
digested and gel-purified pMG211 vector yielded plasmid pMG211_sfp.
sfp_NdeI_f GAT ATA CAT ATG AAG ATT TAC GGA ATT TAT ATG GAC CG
sfp_XhoI_r GTG CTC GAG AAG CTC TTC GTA CGA GAC CAT TGT G
TycAβpY and TycAβF. To construct plasmids encoding selected TycA variants containing mutations
required for β-amino acid incorporation, the C-terminal His6-tag of pSU18 was switched to the
N-terminus in order to have a free C-terminus for interaction with downstream modules. To that end,
NHis_tycA and NHis_tycA_W239S were amplified with primer pair NHis_tycA_EcoRI_f /
11
NHis_tycA_BamHI_r from pSU18_tycA and pSU18_tycA_W239S17, respectively, and spin column
purified. The fragments and plasmid pSU18_tycA were digested with EcoRI / BamHI restriction
endonucleases, gel purified, and ligated to yield plasmids pSU18NHis_tycA and
pSU18NHis_tycA_W239S, respectively. To insert the relevant mutations, tycA was amplified as three
fragments from pSU18NHis_tycA with primer pairs NHis_tycA_EcoRI_f / tycA_A236V_r,
tycA_A236V_f / tycA_CLV_r (Trp239) or tycA_A236V_W239S_f / tycA_CLV_r (Ser239), and
tycA_CLV_f / NHis_tycA_BamHI_r. The PCR amplified fragments were gel purified and assembled by
overlap PCR with primer pair NHis_tycA_EcoRI_f / NHis_tycA_BamHI_r. The assembly products and
plasmid pSU18NHis_tycA were digested with EcoRI / BamHI restriction endonucleases, gel purified, and
ligated to yield plasmids pSU18NHis_tycA_VWCLV (for production of TycAβF) and
pSU18_NHis_tycA_VSCLV (for production of TycAβpY), respectively.
NHis_tycA_EcoRI_f AAT GCA GAA TTC ATT AAA GAG GAG AAA TTA ACC ATG CAT CAC CAT CAC
CAT CAC TCC GGA AGA TCT GTA GCA AAT CAG GCC AAT CTC AT
NHis_tycA_BamHI_r GC AAT TGG ATC CTA GCG CAG TGT ATT TGC AAG CAA TTC GAA GAT
tycA_A236V_f CC AGC ATG TCG TTC GAC GTG TCC GTT TGG GAA ATG TTC ATG GCT TTG
CTG TCT GG
tycA_A236V_W239S_f CC AGC ATG TCG TTC GAC GTG TCC GTT AGC GAA ATG TTC ATG GCT TTG
CTG TCT GG
tycA_A236V_r C GTC GAA CGA CAT GCT GG
tycA_CLV_f GCA TAC GGC CCG ACG GAA TGC CTG GTG GCG ACG ATC TGG GAA GCC
tycA_CLV_r TTC CGT CGG GCC GTA TGC
TycAβpY-AN and TycAβF-AN. Plasmids encoding the C-terminally truncated A domains TycAβpY-AN and
TycAβF-AN were constructed by PCR amplification with primer pair tycA-N_f / tycA-N_r and the
templates pSU18NHis_tycA_VSCLV and pSU18NHis_tycA_VWCLV, respectively. The PCR products
were gel purified, phosphorylated with T4 polynucleotide kinase, and ligated with T4 ligase to yield
plasmids pSU18NHis_tycA-AN_VSCLV and pSU18NHis_tycA-AN_VWCLV, respectively.
tycA-N_f TAG GAT CCA GAT CTC ATC ACC ATC
tycA-N_r GAT TCT GCC GAG AAA CTC GAT C
TycB1-SrfTEP26G. To equip TycB1 with the SrfC thioesterase domain containing point mutation P26G for
peptide offloading (SrfTEP26G)33, the srfC gene was cloned from Bacillus subtilis genomic DNA (ATCC
21332, surfactin producer) into the pTrc99a37 vector. Genomic DNA was isolated and purified by
isopropanol precipitation as previously described57 and served as a template to PCR amplify the srfC gene
with primer pair srfC_KpnI_f / srfC_XbaI_r. The PCR product was spin column purified and digested
12
with KpnI and XbaI restriction endonucleases. A second fragment was PCR amplified from
pTrc99a_tycB1 with primer pair pTrc99a_f / pTrc99a_r, spin column purified, and digested with MluI
and KpnI restriction endonucleases. Both fragments were ligated into the MluI / XbaI-digested and gel-
purified pTrc99a vector to yield plasmid pTrc99a_srfC with a C-terminal His6-tag.
The P26G33 mutation was introduced by single primer mutagenesis with primer P26G followed by
addition of restriction endonuclease DpnI. After 2 h incubation at 37 °C, DNA was spin column purified
and directly transformed into electrocompetent HM007954 cells. The resulting plasmid
pTrc99a_srfC_P26G served as a template for PCR amplification of the TE domain with primer pair
srfC_TE_f / srfC_TE_BamHI_r. Simultaneously, tycB1 was PCR amplified with primer pair
tycB1_TE_KpnI_f / tycB1_TE_r and both fragments were gel purified and assembled by overlap PCR
with primer pair tycB1_KpnI_f / srfC_TE_BamHI_r. The assembled fragment and pTrc99a_srfC were
digested with KpnI / BamHI restriction endonucleases, gel purified, and ligated to yield plasmid
pTrc99a_tycB1_srfTE_P26G.
srfC_KpnI_f G AAA GGT ACC ATG TCT CAA TTT AGC AAG GAT CAG G
srfC_XbaI_r T GAC TCT AGA TTA AGC TTA GTG ATG GTG ATG GTG ATG AGA
TCT GGA TCC TGA AAC CGT TAC GGT TTG TGT ATT AAG
pTrc99a_f CGG CGA TTA AAT CTC G
pTrc99a_r G TCA GGT ACC TTT CCT GTG TGA AAT TGT TAT CC
P26G ATT TTC GCA TTT CCG GGG GTC TTG GGC TAT GGC CT
srfC_TE_f GGG GGC TCT GAT GGC TTG CAG GAT GTA
srfC_TE_BamHI_r GGT GAT GAG ATC TGG ATC CTG AAA CCG TTA CGG
tycB1_TE_KpnI_f AGG AAA GGT ACC ATG AGT GTA TTT AGC AAA GAA CAA GTT CAG G
tycB1_TE_r TAC ATC CTG CAA GCC ATC AGA GCC CCC TTC CAC ATA CGC TGC CAG CGC
TTG AAT CGT
GrsB. Plasmid pTrc99a_grsB for the production of C-terminally His6-tagged GrsB was constructed by
Gibson assembly of 3 fragments (pTrc99a, grsB_1, and grsB_2) containing 20 bp overlaps each using a
Gibson Assembly cloning kit (NEB) according to manufacturer specifications. Fragment pTrc99a was
PCR amplified from pTrc99a_tycB137 with primer pair pTrc99a_BamHI_f / pTrc99a_XhoI_r and gel
purified. Fragments grsB_1 and grsB_2 were PCR amplified from Aneurinibacillus migulanus genomic
DNA (ATCC 9999, gramicidin S producer) with primer pairs grsB_1_f / grsB_1_r and grsB_2_f /
grsB_2_r, respectively. A. migulanus genomic DNA was isolated and purified by isopropanol
precipitation as previously described57. To eliminate a second start site in grsB (Met3574), two
overlapping fragments were PCR amplified from pTrc99a_grsB with primer pairs grsB_NheI_f /
13
grsB_M3574L_r and grsB_M3574L_f / grsB_BamHI_r. The amplified fragments were gel purified,
assembled by overlap PCR with primer pair grsB_NheI_f / grsB_BamHI_r, and introduced into
pTrc99a_grsB via restriction sites NheI / BamHI to yield plasmid pTrc99a_grsB_M3574L.
pTrc99a_BamHI_f A AAA GGA TCC AGA TCT CAT CAC CAT CAC C
pTrc99a_XhoI_r A TGT ACT CAT CTC GAG TTT CCT GTG TGA AAT TGT TAT CC
grsB_1_f G AAA CTC GAG ATG AGT ACA TTT AAA AAA GAA CAT GTT CAG G
grsB_1_r CCA TTC GTT TTG CCA TAC AGC
grsB_2_f GCT GTA TGG CAA AAC GAA TGG
grsB_2_r G ATG AGA TCT GGA TCC TTT TAC TAC AAA TGT CCC TTG TAG TAT CTG
grsB_NheI_f G CTT CAG ATG GCT AGC TTT GCC
grsB_M3574L_r CGT ATC ATT GAA CTC AAG TAA GAT TTG TTT CTT CT
grsB_M3574L_f AG AAG AAA CAA ATC TTA CTT GAG TTC AAT GAT ACG
grsB_BamHI_r AGA TCT GGA TCC TTT TAC TAC AAA TGT CCC TTG TAG TAT CTG
High-Throughput Adenylation and Thioesterification Assay
Buffers and media:
SD-CAA, pH 6 D-glucose (Sigma, 20 g)
yeast nitrogen base without amino acids (Sigma, 6.7 g)
casamino acids (BD, 5 g)
Na2HPO4·7H2O (Acros, 10.19 g) and NaH2PO4·H2O (ABCR, 8.56 g)
The components were dissolved in H2O (1 L) and filter sterilized.
SG-CAA, pH 6 analogous to SD-CAA but with D-galactose (Sigma, 20 g) instead of D-glucose
PMB, pH 7.4 NaH2PO4 (ABCR, 7.2 mM)
Na2HPO4 (Acros, 40 mM)
NaCl (Merck, 137 mM)
KCl (Sigma, 2.7 mM)
MgCL2 (Sigma, 1 mM)
The components were dissolved in H2O and filter sterilized. Directly before use
the buffer was supplemented with BSA (Sigma,1 mg/mL).
Yeast surface display of minimal NRPS modules was performed according to Boder and Wittrup54. Dense
cultures of EBY100 cells transformed with the display plasmids were diluted to an OD600 of 0.1 in SD-
CAA medium, grown to an OD600 of 1 at 30 °C and 250 rpm, and incubated at 20°C and 250 rpm for 16 h
14
in SG-CAA medium (to an OD600 of ~2). Induced cells (180 μL for single variants, 900 μL for libraries)
were collected, pelleted by centrifugation (2 min at 3,000 x g and 4 °C), and the supernatant was
removed. After washing with PMB (180 μL), cells were pelleted by centrifugation, resuspended in PMB
(50 μL) supplemented with recombinant Sfp (4 μM) and CoASH (Thermo Fisher, 500 μM), and
incubated at RT for 15 min to load the ppant arm. For amino acid loading, cells were pelleted by
centrifugation and resuspended in PMB (180 μL) containing ATP (Sigma, 100 μM) and the appropriate
amino acid. The cell suspension was incubated at RT and amino acid loading was stopped by
centrifugation and removal of the supernatant. Subsequently, an azide-alkyne Huisgen cycloaddition26,27
was used to label cells presenting “clickable” amino acids. Cells were incubated with an azide-PEG3-
biotin conjugate (Sigma, 20 μM) in PMB (50 μL) and the reaction was started by addition of freshly
mixed CuSO4 (Sigma, 100 μM), bathophenanthrolinedisulfonic acid (ABCR, 200 μM)58, and L-ascorbic
acid (ABCR, 1 mM, freshly prepared) at 4 °C for 2 h. The reaction was stopped by centrifugation and
removal of the supernatant, followed by washing with PMB (180 μL). The cells were pelleted by
centrifugation, resuspended in PMB (20 μL), and incubated with monoclonal mouse anti-c-myc antibody
9E10 (Roche, 250 ng/μL). After incubation at 4 °C for 30 min, the cells were pelleted by centrifugation,
resuspended in PMB (20 μL), and labelled with goat anti-mouse IgG-FITC antibody (Sigma, 50 ng/μL)
and a streptavidin-R-phycoerythrin conjugate (Thermo Fisher, 50 ng/μL) at 4 °C for another 30 min.
After labelling, the cells were pelleted by centrifugation and washed with PMB (3 x 180 μL). Labelled
cells were resuspended in PMB and analyzed on a LSRFortessa (BD) or sorted (at ∼2000-5000 events/s)
using a FACSAria III (BD) at the Flow Cytometry Core Facility of ETH Zurich. Data was analysed using
the FlowJo software (LLC).
The total number of yeast cells displaying constructs typically varied between ~30%-50% depending
on the time of induction. Cell survival was tested by plating a defined number of sorted cells on SD-CAA
plates and was >50%, where cells with more display were less viable.
The yeast library was enriched in cells encoding TycA variants that selectively load O-propargyl-β-
Tyr over three consecutive FACS rounds. In a first round, the library was screened for loading of racemic
O-propargyl-β-Tyr (400 μM, RT, ∼2 min) and doubly-labelled yeast cells encoding active TycA variants
were sorted into SD-CAA medium supplemented with chloramphenicol (20 μg/mL). A total of 107 cells
was processed (corresponding to a ~10-fold oversampling) and the top 0.5% of the library, which
exhibited FITC and high R-PE fluorescence, was collected by FACS. Sorted cells were regrown at 30 °C
and directly subjected to two analogous rounds of FACS and regrowth with increasingly stringent
conditions for amino acid loading (second round: 12.5 μM O-propargyl-(S)-β-Tyr, RT, ~2 min; third
round: 1 μM O-propargyl-(S)-β-Tyr in the presence of 1 mM competing 4-methoxy-L-Phe (Bachem), RT,
15
~2 min). In both sorting rounds the top 1% doubly-labelled cells were isolated. The enriched library
obtained after three rounds of FACS was plated out onto SD-CAA medium for analysis of individual
variants.
Protein Production
Modified Studier medium59 “ZYM-G” was used for all protein production:
ZY: tryptone (Merck, 1% m/m)
yeast extract (Merck, 0.5% m/m)
50 x M solution (20 mL/L): Na2HPO4 (Merck, 25 mM)
KH2PO4 (Merck, 25 mM)
NH4Cl (Merck, 50 mM)
Na2SO4 (Merck, 5mM)
50 x G solution (20 mL/L): glycerol (Sigma, 50% v/v)
500 x Mg2+ solution (2 mL/L): MgSO4 (Merck, 1 M)
The ZY component was autoclaved, while M, G, and Mg2+ components were sterile filtered (TPP,
0.22 µm) before use (note: LB Miller broth (Merck) could be substituted for ZY with no discernible
difference.) Isopropyl-β-D-thiogalactopyranoside (IPTG), ampicillin (amp), and chloramphenicol (cam)
were obtained from Apollo scientific.
HM007954 cells transformed with expression plasmids were taken from glycerol cell stocks stored
at -80 °C and grown overnight at 37 °C in LB Miller broth (5 mL) containing the respective antibiotic.
The following morning, ZYM-G (800 mL in a 2 L baffled flask) containing the respective antibiotic was
inoculated with the overnight culture (1/500 v/v) and shaken at 180 rpm and 37 °C until an OD600 of 5-7
was reached, at which point the cultures were cooled to 20 °C. After reaching 20 °C, cell cultures were
induced with IPTG (300 μM) and shaken at 180 rpm and 20 °C for ~18 h. Cells were pelleted at 5,000 x g
and 4 °C for 15 minutes, transferred to 50 mL falcon tube(s), and frozen at -20 °C.
Protein Purification
Imidazole, NaCl, HEPES, and Tris were obtained from Merck. pH of all buffers was adjusted using a
WTW bench pH/mV meter (routine meter pH 526) calibrated according to manufacturer specifications.
Cell pellets were thawed and suspended in lysis buffer [3 mL / g cells, Tris (50 mM), NaCl (500 mM),
glycerol (10% v/v), pH 7.4] via vortex. For lysis, the cell suspension was treated by addition of lysozyme
(Sigma, 2 mg/mL), polymyxin B (Apollo, 2 mg/mL), and DNase I (Sigma, ~1 mg total) on ice for 30 min
16
before sonication (Dr. Heilscher, UP 200s sonic dismembrator, total sonication time of 6 min at 100%
power with care taken to keep internal temperature <15 °C). Cellular debris was pelleted in a precooled
(4 °C) centrifuge at 30,000 x g for 20 min and the supernatant was applied to Ni-NTA resin (Qiagen,
1 mL / 2 g cells, pre-equilibrated with five column volumes of lysis buffer). Wash buffer [5 volumes, Tris
(50 mM), NaCl (500 mM), imidazole (20 mM), glycerol (10% v/v), pH 7.4] was added and the column
was gently pressurized with a syringe. The enzyme of interest was eluted with elution buffer [15 mL, Tris
(50 mM), NaCl (500 mM), imidazole (300 mM), glycerol (10% v/v), pH 7.4] with gentle syringe
pressure.
C-terminally truncated A domains (for crystallization) and α-TycA variants (for reduction of
endogenous amino acid background in the 32P-PPi/ATP exchange assay) were buffer exchanged to FPLC
buffer [Tris (20 mM), NaCl (20 mM), glycerol (5% v/v), pH 8.0] using centrifugal filter units (Merck,
Amicon Ultra-15) and purified by anion-exchange chromatography (GE Healthcare, MonoQ 10/100,
linear gradient from 0.05 to 0.5 M NaCl) (Supplementary Fig. 3). For crystallography, the pooled protein
fraction was buffer exchanged to gel-filtration buffer [Tris (20 mM), NaCl (150 mM), pH 8.0] using
centrifugal filter units (Amicon Ultra-15), further purified to homogeneity by gel-filtration
chromatography (GE Healthcare, Superose 12), and concentrated to 27 mg/mL.
All other proteins were buffer exchanged to storage buffer [HEPES (50 mM), NaCl (150 mM),
glycerol (10% v/v), pH 7.4] and aliquoted. Purified proteins were immediately flash frozen in liquid N2
and stored at -80 °C until use. Protein concentration was determined using a Nanodrop 2000
spectrophotometer (Thermo Fisher) corrected by the calculated extinction coefficient (ProtParam,
http://web.expasy.org/protparam/). Protein purity was assessed by SDS-PAGE using a Phast system and
7.5% gels (GE), according to manufacturer specifications (Supplementary Fig. 3).
Identification of a Second Start Site in grsB
Initial attempts to produce wild-type GrsB in E. coli HM007937 predominantly afforded a truncated
protein after Ni-NTA purification (SDS-PAGE app. MW ~100 kDa). To identify this product and
improve GrsB production, the SDS-PAGE band was cut out of the gel and analyzed by Edman
degradation and MALDI-TOF MS (Functional Genomics Center, University of Zurich). The sequence of
the N-terminus (MLEFN) and mass of the protein (MALDI-TOF: 101,331 Da) suggested that the
observed product was produced from an alternative start site at Met3574. The mutation M3574L
eliminated production of the 100 kDa side product and GrsB activity was confirmed by in vitro
production of gramicidin S (Supplementary Fig. 7).
17
Pyrophosphate Exchange Assay
Adenylation kinetics were determined with a pyrophosphate exchange assay as described in Kries et al.17.
Crystallization and Structure Determination of β-A Domains
Crystallization of the full-length engineered β-A domains (Met1-Glu548) was attempted but did not yield
diffracting protein crystals. Accordingly, the respective C-terminally truncated TycA variants (Met1-
Ile417, TycAβpY-AN and TycAβF-AN) were produced and crystallized as previously reported29. A Phoenix
crystallization robot (Art Robbins Instruments) was used to set up sitting-drop vapour-diffusion
experiments in Intelli-Plates R96-3 LV (Hampton Research). Initial crystallization attempts were carried
out at 4 °C with conditions identified using the JCSG+ Suite (Qiagen) and Crystal Screen 1 and 2
(Hampton Research), and were later refined by grid screens with varying pH and precipitant
concentrations. Well-diffracting TycAβpY-AN and TycAβF-AN crystals were obtained with 27 mg/mL
enzyme at 4 °C in crystallization buffer [Bis-Tris (100 mM), (NH4)2SO4 (200 mM), PEG3350 (Sigma
Aldrich, 25% (v/v)), pH 5.5] containing 2 mM ligand 5 or 6, respectively, using the sitting-drop vapour-
diffusion method. The crystals were transferred into reservoir solutions with 20% (v/v) glycerol as
cryoprotectant and flash cooled at -173 ˚C in a N2 stream. X-ray diffraction data sets were collected at the
X06SA macromolecular crystallography beamline of the Swiss Light Source (Paul Scherrer Institute,
Villigen, Switzerland) using an EIGER X 16M detector and wavelengths of 1.0000 Å.
The diffraction data for TycAβpY and TycAβF were processed and scaled using the XDS60 program
package. Initial phases were determined by molecular replacement with Phaser61 using the structure of
the GrsA A domain (PDB 1AMU)28 as a search model. The structure was modified manually with Coot62
and refined with PHENIX63. The final crystal data and intensity statistics are summarized in
Supplementary Table 2. The final model of TycAβF-AN (PDB 5N82) consists of a single chain containing
residues 20-180 and 183-417, ligand 5, and 328 molecules of water. The final model of TycAβpY-AN
(PDB 5N81) consists of two nearly identical chains (A and B, RMSD of 0.4 Å) containing residues 19-
179 and 183-417, ligand 6, and 848 molecules of water. A structural similarity search was performed
using Dali64. The cavity volumes were calculated with CASTP (http://cast.engr.uic.edu/cast/). All
crystallographic figures were prepared with PyMOL (DeLano Scientific, http://www.pymol.org).
Dipeptide Synthetase Reactions
To facilitate release of βα-dipeptides, TycB1 was fused to the robust surfactin TE domain65 containing
the P26G33 mutation that is known to promote hydrolysis. Surprisingly, the bi-modular synthetases
18
consisting of the β-TycA variants and TycB1-SrfTEP26G predominantly catalysed formation of the cyclic
βα-dipeptide.
Dipeptide synthetase reactions were performed at 37 °C (water bath) in a volume of 250 μL, and
initiated by the addition of L-Phe, O-propargyl-L-Tyr, O-propargyl-(S)-β-Tyr, (S)-β-Phe, or a mixture of
all four. All reactions contained TycB1-SrfTEP26G (2 μM) and one of the four TycA variants (TycAF,
TycApY, TycAβpY, or TycAβF, 2 μM). Reactions were performed in triplicate and monitored at 15, 30, and
45 min time points where 50 μL of the reaction mixture was aliquoted into ice-cold MeOH (150 μL),
vortexed, and clarified by centrifugation (21,000 x g, 5 min, 4 ˚C). Product formation was quantified by
HPLC (Ultimate 3000, Dionex) using a Reprosil Gold 120 C-18 column (100 x 2.1 mm, 3 μm): 5 μL
injection, monitoring 220 nm, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flow rate =
0.75 mL/min, 0-0.2 min = 5% B, 0.2-4.75 min ramp to 40% B, 4.75-5 min ramp to 100% B, 5-5.5 min =
100% B, 5.5-5.6 min ramp to 5 % B, 5.6-7 min reequilibration = 5%. Calibration curves generated from 5
concentrations of authentic standards (9, 10, and DKPs17, from 4% to 200% conversion) were linear and
used to quantify reaction progress.
Reaction conditions: Bis-Tris propane (100 mM), NaCl (100 mM), MgCl2 (10 mM), ATP (5 mM),
L-Pro (1 mM), L-Phe, O-propargyl-L-Tyr, O-propargyl-(S)-β-Tyr, (S)-β-Phe, or a combined mixture of all
four (1 mM), pH = 9.
For total turnover number (TTN) determination of dipeptide synthetases, three concentrations were
examined [amino acids (5 mM, 10 mM, 15 mM) and corresponding ATP (25 mM, 50 mM, 75 mM)]
under otherwise identical conditions, allowing theoretical maximum TTNs of 2500, 5000, and 7500,
respectively.
Pentapeptide Synthetase Reactions
Enzymatic reactions for in vitro pentapeptide and gramicidin S production were performed and analyzed
as described for the dipeptide synthetase and reaction progress was monitored after 20, 40, and 60 min.
All reactions contained TycAF or TycAβF (1 μM) with an excess of GrsB (4 μM).
Product formation was quantified by HPLC (Ultimate 3000, Dionex) using a Kinetex XB-C18 column
(100 x 4.6 mm, 2.6 μm): 10 μL injection, monitoring 220 nm, solvent A = H2O + 0.1% TFA, solvent B =
MeCN + 0.1% TFA, flowrate = 1.5 mL/min, 0-1 min = 5% B, 1-4 min ramp to 40% B, 4-5.5 min ramp to
95% B, 5.5-7 min = 95% B, 7-7.1 min ramp to 5 % B, 7.1-8.5 min reequilibration = 5% or by LC-MS
(Waters H-class UPLC/SQD-2) using an Acquity UPLC BEH C-18 column (50 x 2.1 mm, 1.7 μm), 1 µL
injection, monitoring ESI+ for [M+2H]2+ = 295.5±2 m/z or 572±2 m/z, solvent A = H2O + 0.1% TFA,
solvent B = MeCN + 0.1% TFA, flow rate = 1 mL/min, initial conditions = 5% B, 0-1.5 min ramp to 80%
19
B, 1.5-2 min ramp to 100% B, 2-2.2 min = 100% B, 2.2-2.3 min ramp to 5 % B, 2.3-3 min reequilibration
= 5%. Calibration curves generated from 5 concentrations of the authentic standards [(S)-β-Phe-L-Pro-L-
Val-L-Orn-L-Leu was synthesized by standard FMOC SPPS, gramicidin S was purchased from Sigma]
were linear and used to quantify reaction progress.
Reaction conditions: 100 mM Bis-Tris propane, 100 mM NaCl, 10 mM MgCl2, 20 mM ATP, 1.5 mM
L-Pro, 1.5 mM L-Val, 1.5 mM L-Orn, 1.5 mM L-Leu, and 1 mM L-Phe or (S)-β-Phe, pH = 8.
In Vivo Pentapeptide Production
LB (5 mL) containing ampicillin (250 μg/mL) and chloramphenicol (37.5 μg/mL) was inoculated with
HM007937 cells transformed with plasmids pTrc99a_grsB_M3574L and pSU18NHis_ tycA_VWCLV
and grown at 37 °C, 250 RPM until an OD600 = 1 was reached.
Modified Studier medium “ZYM-G” (30 mL in a 300 mL flask, as described in section Protein
Production) supplemented with (S)-β-Phe (1 mM), L-Pro (1 mM), L-Val (1 mM), L-Orn (1 mM), L-Leu
(1 mM), ampicillin (250 μg/mL), and chloramphenicol (37.5 μg/mL) was inoculated with the starter
culture (1/250 v/v) and incubated at 37 °C, 280 RPM. Upon reaching OD600 = 1.75, the cultures were
cooled (20 °C, 280 RPM) and induced with IPTG (100 μM).
Culture medium was sampled at 24 h and 48 h (100 μL culture into 900 μL MeOH), vortexed
vigorously, and clarified by centrifugation (21,000 x g, 5 min, RT).
Product formation was quantified by LC-MS (Waters H-class UPLC/SQD-2) using an Acquity UPLC
BEH C-18 column (50 x 2.1 mm, 1.7 μm), 500 nL injection, monitoring ESI+ for [M+2H]2+ =
295.2±2 m/z, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flow rate = 0.5 mL/min,
initial conditions = 5% B, 0-4 min ramp to 30% B, 4-5 min ramp to 100% B, 5-6 min = 100% B, 6-6.5
min ramp to 5 % B, 6.5-7 min reequilibration = 5%. A calibration curve generated from 5 concentrations
of the authentic standard (1-50 μM) was linear and used to calculate the titre of (S)-β-Phe-L-Pro-L-Val-L-
Orn-L-Leu (11).
20
Supplementary Figures
Supplementary Figure. 1. A high-throughput assay for adenylation and thioesterification. a, Flow
cytometry of yeast EBY100 cells54 displaying minimal TycAF (lacking the E domain) loaded with an
equimolar mixture (20 μm each) of L-Phe and O-propargyl-L-Tyr. Yeast cell surface display was
monitored by immunofluorescent FITC-labelling of a C-terminal c-myc tag and R-PE-conjugated
streptavidin was used to label loading of O-propargyl-L-Tyr and conjugation to N3-PEG3-biotin by an
azide-alkyne Huisgen cycloaddition26,27. A typical sorting gate is indicated by the blue square (double
label cells, 1.8% of total population), whereas the green square highlights single label cells (29.8% of
total population). b, Flow cytometry of identically treated yeast EBY100 cells displaying minimal TycApY
(7.6% single label, 26.9% double label).
21
Supplementary Figure 2. Sequences of selected O-propargyl-(S)-β-Tyr-specific TycA variants.
Sequence alignment of TycAF with ten variants obtained after three rounds of FACS screening for loading
of O-propargyl-(S)-β-Tyr. Engineered positions are highlighted in grey.
22
Supplementary Figure 3. TycA purification and characterization. a, SDS-PAGE analysis of all four
TycA variants after NiNTA affinity chromatography. b, Chromatogram of MonoQ-purified TycAβF
(black line: absorption at 280 nm, grey line: absorption at 260 nm, thin black line: conductivity). The
protein elutes in the fraction centred around 3500 s; the peak at 5200 s absorbs strongly at 260 nm and
contains no protein. c, SDS-PAGE analysis of NiNTA- and FPLC-purified TycAβF. For further
confirmation of protein identity, both samples were sent to the Functional Genomics Center Zurich for
mass determination (calculated for holo-TycAβF: 124,147 Da, found: 124,150 Da (NiNTA), 124,144 Da
(FPLC)).
23
Supplementary Figure 4. Active sites of TycAβpY-AN and TycAβF-AN. a, Stereoview of TycAβpY-AN in
complex with ligand 6 (yellow sticks) and b, Stereoview of TycAβF-AN in complex with ligand 5 (yellow
sticks). The Fo-Fc omit maps for the ligands are contoured at 3 σ. c, Binding pocket of TycAβF-AN with
bound ligand 5 (yellow spheres) from two different angles. The side chains of active site residues that
contact the ligand are shown as sticks. Hydrogen bonding interactions are indicated by grey dashes.
Positions that were engineered in this study are highlighted in red.
24
Supplementary Figure 5. Structural rationale for the α/β-switch. a, Overlay of the β13β14 strand-
loop-strand segments from TycAβF (grey), GrsA28 (cyan), and VinN29 (magenta). The engineered loop
positions in TycAβF are highlighted in red. b, Comparison of the binding modes of L-Phe (magenta
spheres) and (S)-β-Phe (yellow spheres) at the active sites of GrsA (cyan) and TycAβF (grey),
respectively. In both cases, the residues targeted for mutagenesis in this study (transparent surfaces)
bracket the side chain of the amino acid substrate and clamp it in place. Note the change in orientation of
the aryl group of (S)-β-Phe compared to L-Phe.
25
Supplementary Figure 6. β-Amino acid binding mode. Superimposed structures of VinN29 (magenta)
and TycAβF-AN (grey cartoon with green sticks, PDB 5N82) with their respective ligands (2S,3S)-3-
methylaspartate (black) and 5 (yellow, showing only the β-amino acid moiety of the ligand).
26
Supplementary Figure 7. In vitro formation of pentapeptide 11 and gramicidin S. a, LC-MS
chromatograms of authentic pentapeptide 11 and an exemplary in vitro reaction catalysed by TycAβF and
GrsB in the presence of (S)-β-Phe. b, LC-MS chromatograms for authentic gramicidin S and an
exemplary in vitro reaction of TycAF and GrsB with L-Phe. Peak ‘*’ corresponds to the
L-Phe-pentapeptide intermediate. c, Kinetics of pentapeptide 11 formation by TycAβF/GrsB. d, Kinetics of
gramicidin S formation by TycAF/GrsB. Data points represent the mean ± standard deviation, where n ≥
3. Three independent batches of GrsB were used for data collection (TycAβF and TycAF in excess). TIC:
Total ion current, EIC: Elected ion current.
27
Supplementary Figure 8. In vivo formation of pentapeptide 11. a, (S)-β-Phe-containing pentapeptide
11. b, LC-MS chromatograms of aliquots from the culture medium alone, crude E. coli cultures 48 h after
induction, and an authentic pentapeptide 11 standard. c, Determination of pentapeptide 11 titres in E. coli
cultures producing TycAβF and GrsB and supplemented with (S)-β-Phe 24 h (orange, 56±5 mg/L) and 48
h (blue, 120±20 mg/L) after induction by comparison to authentic standards (black dots). Data are
reported as the mean ± standard deviation, where n = 3. TIC: Total ion current, EIC: Elected ion current.
28
Supplementary Figure 9. Active site sequence alignments of representative A domains. a,
Specificity-determining binding pocket residues66,67 of the β-Phe-activating A domains TycAβF, AdmJ,
and HitB and the homologous A domains of GrsA and VinN, which activate L-Phe and (2S,3S)-3-
methylaspartate, respectively. Engineered positions in TycAβF are highlighted with a grey background.
Structures for AdmJ and HitB are not available, so the amino acid identities at the positions denoted with
an asterisk were determined using a homology model generated with the SWISS-MODEL server68. b,
Sequence alignment showing the engineered β13β14 strand-loop-strand. The 328-331 segment targeted
for mutagenesis is highlighted in grey. Loop compositions of AdmJ and HitB were determined based on
the SWISS-MODEL homology models.
29
Supplementary Tables
Supplementary Table 1. Adenylation steady-state parameters. Michaelis-Menten parameters for the
four TycA variants determined with a 32P-PPi/ATP exchange assay31. Data represent the average of three
separate measurements with different batches of protein.
Amino acid
substrate
Kinetic
parameters TycAF TycApY TycAβpY TycAβF
L-Phe
kcat (min-1) 142±34 95±39 n.d.* 29±4
KM (mM) 0.013±0.001 2.3±1.3 n.d.* 2.6±0.3
kcat / KM
(min-1mM-1) 11,100±2,600 39±11 0.009±0.007 11±1
O-propargyl-
L-Tyr
kcat (min-1) n.d.* 132±21 n.d.* n.d.*
KM (mM) n.d.* 0.6±0.2 n.d.* n.d.*
kcat / KM
(min-1mM-1) 0.5±0.2 228±87 0.6±0.3 0.76±0.09
O-propargyl-
(S)-β-Tyr
kcat (min-1) n.d.* 0.6±0.4 31±4 n.d.*
KM (mM) n.d.* 1.8±0.9 0.19±0.02 n.d.*
kcat / KM
(min-1mM-1) 0.12±0.07 0.5±0.6 167±31 0.5±0.1
(S)-β-Phe
kcat (min-1) 1.7±0.5 1.4±0.4 n.d.* 71±8
KM (mM) 0.03±0.01 4.7±1.5 n.d.* 0.030±0.005
kcat / KM
(min-1mM-1) 54±14 0.2±0.1 4.2±0.6 2,400±500
*n.d.: not determined due to the absence of substrate saturation
30
Supplementary Table 2. Data collection, phasing, and refinement statistics.
TycAβF-AN TycAβpY-AN
Data collection
Space group P212121 P212121
Cell dimensions
a, b, c (Å) 59.6, 60.4, 123.8 59.6, 60.2, 247.8
Resolution (Å) 50.0-1.7 (1.81-1.71)* 50.0-1.6 (1.63-1.60)*
Rmerge (%) 6.5 (65.3) 6.5 (75.7)
I / σI 19.3 (2.9) 16.3 (2.3)
Completeness (%) 98.9 (97.2) 99.9 (100.0)
Redundancy 6.8 (6.9) 6.8 (7.0)
Refinement
Resolution (Å) 43.2-1.7 43.2-1.6
No. reflections 48726 118611
Rwork / Rfree (%) 17.1/20.0 17.6/20.1
No. atoms
Protein 3112 6205
Ligand/ion 72 119
Water 328 848
B-factors
Protein 24.9 20.7
Ligand/ion 24.4 21.9
Water 33.7 30.9
R.m.s. deviations
Bond lengths (Å) 0.006 0.006
Bond angles (°) 0.918 0.883
* One crystal was used for data collection; values in parentheses are for highest-resolution shell.
31
Supplementary Table 3. Crystal data and structure refinement for compound 9.
Empirical formula C17H18N2O3 Formula weight 298.33 Temperature/K 100.0(1) Crystal system monoclinic Space group P21
a/Å 10.53110(10) b/Å 6.79350(10) c/Å 10.90170(10) α/° 90 β/° 112.6870(10) γ/° 90
Volume/Å3 719.593(15) Z 2
ρcalcg/cm3 1.377 μ/mm-1 0.778 F(000) 316
Crystal size/mm3 0.233 × 0.121 × 0.052 Radiation CuKα (λ = 1.54184)
2Θ range for data collection/° 8.792 to 158.45 Index ranges -13 ≤ h ≤ 13, -7 ≤ k ≤ 8, -13 ≤ l ≤ 13
Reflections collected 56301 Independent reflections 2972 [Rint = 0.0307, Rsigma = 0.0112]
Data/restraints/parameters 2972/2/202 Goodness-of-fit on F2 1.045
Final R indexes [I>=2σ (I)] R1 = 0.0266, wR2 = 0.0705 Final R indexes [all data] R1 = 0.0266, wR2 = 0.0706
Largest diff. peak/hole / e Å-3 0.18/-0.19 Flack parameter -0.01(4)
32
Supplementary Table 4. Crystal data and structure refinement for compound 10.
Empirical formula C14H16N2O2 Formula weight 244.29 Temperature/K 100.0(1) Crystal system orthorhombic Space group P212121
a/Å 7.19770(6) b/Å 9.65236(5) c/Å 18.11426(9) α/° 90 β/° 90 γ/° 90
Volume/Å3 1258.485(14) Z 4
ρcalcg/cm3 1.289 μ/mm-1 0.707 F(000) 520
Crystal size/mm3 0.121 × 0.076 × 0.043 Radiation CuKα (λ = 1.54184)
2Θ range for data collection/° 9.766 to 158.08 Index ranges -8 ≤ h ≤ 9, -12 ≤ k ≤ 12, -23 ≤ l ≤ 23
Reflections collected 90375 Independent reflections 2704 [Rint = 0.0414, Rsigma = 0.0105]
Data/restraints/parameters 2704/1/167 Goodness-of-fit on F2 1.064
Final R indexes [I>=2σ (I)] R1 = 0.0313, wR2 = 0.0824 Final R indexes [all data] R1 = 0.0322, wR2 = 0.0831
Largest diff. peak/hole / e Å-3 0.50/-0.16 Flack parameter 0.02(4)
33
Supplementary Table 5. DNA sequences encoding the proteins used in this study.
Protein Vector DNA Sequence
Sfp pMG211
ATGAAGATTTACGGAATTTATATGGACCGCCCGCTTTCACAGGAAGAAAATGAACGGTTCATGAC
TTTCATATCACCTGAAAAACGGGAGAAATGCCGGAGATTTTATCATAAAGAAGATGCTCACCGCA
CCCTGCTGGGAGATGTGCTCGTTCGCTCAGTCATAAGCAGGCAGTATCAGTTGGACAAATCCGAT
ATCCGCTTTAGCACGCAGGAATACGGGAAGCCGTGCATCCCTGATCTTCCCGACGCTCATTTCAA
CATTTCTCACTCCGGCCGCTGGGTCATTGGTGCGTTTGATTCACAGCCGATCGGCATAGATATCG
AAAAAACGAAACCGATCAGCCTTGAGATCGCCAAGCGCTTCTTTTCAAAAACAGAGTACAGCGAC
CTTTTAGCAAAAGACAAGGACGAGCAGACAGACTATTTTTATCATCTATGGTCAATGAAAGAAAG
CTTTATCAAACAGGAAGGCAAAGGCTTATCGCTTCCGCTTGATTCCTTTTCAGTGCGCCTGCATC
AGGACGGACAAGTATCCATTGAGCTTCCGGACAGCCATTCCCCATGCTATATCAAAACGTATGAG
GTCGATCCCGGCTACAAAATGGCTGTATGCGCCGCACACCCTGATTTCCCCGAGGATATCACAAT
GGTCTCGTACGAAGAGCTTCTCGAGCACCACCACCACCACCACTAA
TycAF pSU18NHis
ATGCATCACCATCACCATCACTCCGGAAGATCTGTAGCAAATCAGGCCAATCTCATCGACAACAA
GCGGGAACTGGAGCAGCATGCGCTAGTTCCATATGCACAGGGCAAGTCGATCCATCAATTGTTCG
AGGAACAAGCAGAGGCTTTTCCAGACCGCGTTGCCATCGTTTTTGAAAACAGGCGGCTTTCGTAT
CAGGAGTTGAACAGGAAAGCCAATCAACTGGCAAGAGCCTTGCTCGAAAAAGGGGTGCAAACAGA
CAGCATCGTCGGTGTGATGATGGAGAAGTCCATCGAAAATGTCATCGCGATTCTGGCCGTTCTTA
AAGCAGGCGGAGCCTATGTGCCCATCGACATCGAATATCCCCGCGATCGCATCCAATATATTTTG
CAGGATAGTCAAACGAAAATCGTGCTTACCCAAAAAAGCGTCAGCCAGCTCGTGCATGACGTCGG
GTACAGCGGAGAGGTAGTTGTACTCGACGAAGAACAGTTGGACGCTCGCGAGACTGCCAATCTGC
ACCAGCCCAGCAAGCCTACGGATCTTGCCTATGTCATTTACACCTCAGGCACGACAGGCAAGCCA
AAAGGCACCATGCTTGAACATAAAGGCATCGCCAATTTGCAATCCTTTTTCCAAAATTCGTTTGG
CGTCACCGAGCAAGACAGGATCGGGCTTTTTGCCAGCATGTCGTTCGACGCATCCGTTTGGGAAA
TGTTCATGGCTTTGCTGTCTGGCGCCAGCCTGTACATCCTTTCCAAACAGACGATCCATGATTTC
GCTGCATTTGAACACTATTTGAGTGAAAATGAATTGACCATCATCACACTGCCGCCGACTTATTT
GACTCACCTCACCCCAGAGCGCATCACCTCGCTACGCATCATGATTACGGCAGGATCAGCTTCCT
CCGCACCCTTGGTAAACAAATGGAAAGACAAACTCAGGTACATAAATGCATACGGCCCGACGGAA
ACGAGCATTTGCGCGACGATCTGGGAAGCCCCGTCCAATCAGCTCTCCGTGCAATCGGTTCCGAT
CGGCAAACCGATTCAAAATACACATATTTATATCGTCAATGAAGACTTGCAGCTACTGCCGACTG
GCAGCGAAGGCGAATTGTGCATCGGCGGAGTCGGCTTGGCAAGAGGCTATTGGAATCGGCCCGAC
TTGACCGCAGAAAAATTCGTAGACAATCCGTTCGTACCAGGCGAAAAAATGTACCGCACAGGTGA
CTTGGCCAAATGGCTGACGGATGGAACGATCGAGTTTCTCGGCAGAATCGACCATCAGGTGAAAA
TCAGAGGTCATCGCATCGAGCTTGGCGAAATCGAGTCTGTTTTGTTGGCACATGAACACATCACA
GAGGCCGTGGTCATTGCCAGAGAGGATCAACACGCGGGACAGTATTTGTGCGCCTATTATATTTC
GCAACAAGAAGCAACTCCTGCGCAGCTCAGAGACTACGCCGCCCAGAAGCTTCCGGCTTACATGC
TGCCATCTTATTTCGTCAAGCTGGACAAAATGCCGCTTACGCCAAATGACAAGATCGACCGCAAA
GCGTTGCCCGAGCCTGATCTTACGGCAAACCAAAGCCAGGCTGCCTACCATCCTCCGAGAACCGA
GACAGAATCGATTCTCGTCTCCATCTGGCAAAACGTTTTGGGAATTGAAAAGATCGGGATTCGCG
ATAATTTTTACTCGCTCGGCGGAGATTCGATCCAAGCGATCCAGGTCGTGGCTCGTCTGCATTCC
TATCAATTGAAGCTAGAGACGAAAGACTTGCTGAATTACCCGACGATCGAGCAGGTTGCTCTTTT
TGTCAAGAGCACGACGAGAAAAAGCGATCAGGGCATCATCGCTGGAAACGTACCGCTTACACCCA
TTCAGAAGTGGTTTTTCGGGAAAAACTTTACGAATACAGGCCATTGGAACCAATCGTCTGTGCTC
TATCGCCCGGAAGGCTTTGATCCTAAAGTCATCCAAAGTGTCATGGACAAAATCATCGAACACCA
CGACGCGCTCCGCATGGTCTATCAGCACGAAAACGGAAATGTCGTTCAGCACAACCGCGGCTTGG
GTGGACAATTATACGATTTCTTCTCTTATAATCTGACCGCGCAACCAGACGTCCAGCAGGCGATC
GAAGCAGAGACGCAACGTCTGCACAGCAGCATGAATTTGCAGGAAGGACCTCTGGTGAAGGTTGC
CTTATTTCAGACGTTACATGGCGATCATTTGTTTCTCGCAATTCATCATTTGGTCGTGGATGGCA
TTTCCTGGCGCATTTTGTTTGAAGATTTGGCAACCGGATACGCGCAGGCACTTGCAGGGCAAGCG
ATCAGTCTGCCCGAAAAAACGGATTCTTTTCAAAGCTGGTCACAATGGTTGCAAGAATATGCGAA
CGAGGCGGATTTGCTGAGCGAGATTCCGTACTGGGAGAGTCTCGAATCGCAAGCAAAAAATGTGT
34
CCCTGCCGAAAGACTATGAAGTGACCGACTGCAAACAAAAGAGCGTGCGAAACATGCGGATACGG
CTGCACCCGGAAGAGACCGAGCAGTTGTTGAAGCACGCCAATCAGGCCTATCAAACGGAAATCAA
CGATCTGTTGTTGGCGGCGCTCGGCTTGGCTTTTGCGGAGTGGAGCAAGCTTGCGCAAATCGTCA
TTCATTTGGAGGGGCACGGGCGCGAGGACATCATCGAACAGGCAAACGTGGCCAGAACGGTCGGA
TGGTTTACGTCGCAATATCCGGTATTGCTCGACTTGAAGCAAACCGCTCCCTTGTCCGACTATAT
CAAGCTCACCAAAGAGAATATGCGGAAGATTCCTCGTAAAGGGATCGGTTACGACATCTTGAAGC
ATGTGACACTTCCAGAAAATCGCGGTTCCTTATCCTTCCGCGTGCAGCCGGAAGTGACGTTCAAC
TACTTGGGACAGTTTGATGCGGACATGAGAACGGAACTGTTTACCCGCTCACCCTACAGCGGCGG
CAACACGTTAGGCGCAGATGGCAAAAACAATCTGAGTCCTGAGTCAGAGGTGTACACCGCTTTGA
ATATAACCGGATTGATTGAAGGCGGAGAGCTCGTCCTCACATTCTCTTACAGCTCGGAGCAGTAT
CGGGAAGAGTCCATCCAGCAATTGAGCCAAAGTTATCAAAAGCATCTGCTTGCCATCATCGCGCA
TTGCACCGAGAAAAAAGAAGTAGAGCGAACGCCCAGCGATTTCAGCGTCAAAGGTCTCCAAATGG
AAGAAATGGACGATATCTTCGAATTGCTTGCAAATACACTGCGCTAG
NNN: TycAF-AT
NNN: TycAF-AN
GCA: A236, GTG in TycAβpY and TycAβF
TGG: W239, AGC in TycApY and TycAβpY
ACGAGCATTTGC: β13β14 loop, TGCCTGGTG in TycAβpY and TycAβF
TycB1-
SrfTEP26G pTrc99a
ATGAGTGTATTTAGCAAAGAACAAGTTCAGGATATGTATGCGTTGACCCCGATGCAAGAGGGGAT
GCTGTTTCACGCCTTGCTCGACCAAGAGCACAACTCGCATCTGGTACAGATGTCGATTTCGTTGC
AGGGCGATCTTGACGTTGGGCTATTTACGGATAGCCTGCATGTGCTGGTAGAGAGATACGATGTA
TTCCGCACGTTGTTTCTCTATGAAAAGCTGAAGCAGCCTTTGCAAGTTGTCTTGAAGCAACGGCC
TATTCCGATCGAATTTTACGGCTTGTCTGCCTGCGACGAGTCCGAGAAACAACTTCGCTATACGC
AATACAAAAGGGCGGATCAGGAGCGGACGTTTCATCTGGCAAAAGACCCGTTGATGCGGGTCGCC
CTTTTCCAAATGTCCCAGCACGACTACCAGGTCATCTGGAGCTTTCATCACATCCTCATGGACGG
CTGGTGCTTCAGCATTATTTTTGATGACTTGCTTGCCATCTACTTGTCCTTGCAAAACAAGACGG
CACTCTCCCTGGAGCCCGTACAGCCATACAGTCGCTTTATCAACTGGCTGGAAAAACAAAATAAA
CAGGCCGCTCTCAACTATTGGAGCGACTATCTGGAAGCCTATGAACAAAAGACTACCTTGCCGAA
GAAGGAAGCTGCCTTCGCCAAAGCATTTCAACCAACCCAATACCGCTTTTCGCTGAACCGCACCT
TGACCAAGCAGCTCGGGACCATCGCCAGTCAAAATCAAGTGACGCTATCGACGGTGATTCAAACG
ATCTGGGGAGTTCTCCTGCAAAAATACAATGCGGCCCATGATGTGCTGTTCGGCTCTGTTGTATC
CGGACGCCCTACAGACATCGTCGGAATCGACAAAATGGTTGGCTTGTTTATCAATACGATTCCAT
TCCGGGTGCAAGCGAAAGCTGGTCAAACGTTTTCCGAGCTGTTGCAAGCTGTGCACAAAAGAACT
TTGCAATCACAGCCGTATGAGCACGTGCCTTTGTACGACATTCAAACTCAGTCCGTCTTGAAGCA
GGAGCTGATTGACCACCTGCTGGTCATCGAAAATTACCCGCTGGTAGAGGCTTTGCAGAAAAAAG
CATTGAACCAGCAGATCGGCTTCACGATTACTGCTGTGGAAATGTTCGAGCCGACCAATTACGAC
TTGACTGTCATGGTGATGCCAAAAGAAGAGCTTGCCTTCCGTTTTGACTACAATGCGGCTCTGTT
TGACGAACAGGTCGTGCAAAAACTGGCGGGGCACCTCCAACAGATCGCGGATTGCGTGGCAAACA
ATTCGGGAGTCGAGCTTTGCCAGATTCCGTTGCTGACAGAAGCAGAAACTAGCCAGCTGTTGGCA
AAGCGTACGGAAACAGCGGCTGACTATCCTGCCGCAACCATGCACGAGCTGTTTTCGCGGCAGGC
AGAAAAAACGCCTGAGCAAGTGGCGGTAGTCTTCGCGGATCAGCACCTGACGTATCGGGAGCTGG
ATGAAAAATCCAATCAGCTCGCCCGCTTTTTGCGCAAAAAAGGCATTGGCACGGGCAGTCTTGTC
GGCACGCTGCTGGATCGCTCGCTGGACATGATCGTCGGAATCCTCGGCGTCTTGAAGGCAGGCGG
CGCATTTGTGCCGATCGACCCGGAGTTGCCTGCCGAACGAATCGCTTACATGCTGACGCATAGCA
GAGTTCCATTGGTCGTGACGCAAAATCATTTGCGGGCAAAAGTGACCACGCCTACAGAAACAATT
GACATCAACACAGCGGTGATCGGGGAAGAGAGCCGCGCCCCTATCGAATCGCTCAATCAGCCGCA
TGACTTGTTTTACATCATCTATACGTCCGGAACGACAGGGCAACCGAAAGGCGTCATGCTGGAGC
ATCGCAACATGGCGAACCTGATGCGTTTTACGTTTGATCAGACGAACATCGCTTTTCATGAAAAA
35
GTGTTGCAGTATACCACGTGCAGCTTTGATGTTTGCTACCAGGAAATTTTCTCCACGCTGCTATC
CGGGGGCCAGCTCTACCTGATCACGAACGAGCTGAGACGGCATGTGGAAAAGCTGTTTGCTTTCA
TCCAGGAAAAGCAGATCAGCATTTTGTCTCTCCCGGTGTCCTTCCTGAAATTTATTTTTAACGAA
CAAGACTACGCGCAAAGCTTCCCGCGTTGTGTCAAACATATCATCACGGCCGGGGAACAACTCGT
CGTCACACACGAGCTGCAAAAGTATCTGCGCCAGCATCGCGTATTTTTGCACAATCACTACGGCC
CGTCGGAGACGCATGTGGTGACGACATGCACGATGGACCCGGGACAGGCGATACCAGAGCTGCCG
CCCATCGGAAAGCCGATCAGCAACACAGGCATTTACATTTTGGATGAAGGGCTGCAATTGAAGCC
GGAGGGGATCGTCGGGGAGTTGTACATTTCCGGCGCAAACGTAGGAAGAGGGTATTTGCACCAGC
CGGAGCTGACCGCGGAGAAGTTTCTCGACAATCCGTATCAGCCAGGCGAAAGAATGTACCGAACG
GGTGATCTGGCGCGTTGGTTGCCGGATGGCCAGCTCGAATTTTTGGGCCGAATCGACCATCAGGT
AAAAATCAGGGGCCATCGCATCGAGCTGGGAGAGATCGAATCGCGCCTGCTCAACCATCCCGCCA
TCAAGGAAGCGGTGGTTATCGACCGAGCAGACGAGACAGGCGGCAAGTTTTTGTGCGCCTATGTC
GTCCTGCAAAAAGCGCTCAGTGACGAAGAGATGCGGGCATACTTGGCGCAAGCGTTGCCGGAGTA
TATGATCCCTTCCTTTTTCGTGACGCTGGAGCGGATTCCAGTCACGCCGAACGGAAAAACAGACA
GGCGAGCTTTGCCGAAGCCGGAAGGAAGTGCCAAGACGAAAGCGGATTACGTCGCCCCGACGACT
GAGCTGGAACAAAAGCTGGTCGCGATTTGGGAGCAAATTCTTGGCGTGTCGCCGATCGGCATTCA
GGATCATTTTTTCACGCTGGGCGGCCATTCGTTAAAAGCGATTCAGCTCATTTCCCGCATCCAAA
AGGAATGCCAGGCGGATGTCCCGCTGCGCGTCCTGTTTGAGCAACCGACGATTCAAGCGCTGGCA
GCGTATGTGGAAGGGGGCTCTGATGGCTTGCAGGATGTAACGATAATGAATCAGGATCAGGAGCA
GATCATTTTCGCATTTCCGGGGGTCTTGGGCTATGGCCTTATGTACCAAAATCTGTCCAGCCGCT
TGCCGTCATACAAGCTGTGCGCCTTTGATTTTATTGAGGAGGAAGACCGGCTTGACCGCTATGCG
GATTTGATCCAGAAGCTGCAGCCGGAAGGGCCTTTAACATTGTTTGGATATTCAGCGGGATGCAG
CCTGGCGTTTGAAGCTGCGAAAAAGCTTGAGGGACAAGGCCGTATTGTTCAGCGGATCATCATGG
TCGATTCCTATAAAAAACAAGGTGTCAGTGATCTGGACGGACGCACGGTTGAAAGTGATGTCGAA
GCGTTGATGAATGTCAATCGGGACAATGAAGCGCTCAACAGCGAAGCCGTCAAACAAGGCCTCAA
GCAAAAAACACATGCCTTTTACTCATACTACGTCAACCTGATCAGCACAGGCCAGGTGAAAGCAG
ATATTGATCTGTTGACTTCCGGCGCTGATTTTGACATACCGGAATGGCTTGCATCATGGGAAGAA
GCTACAACAGGTGCTTACCGTATGAAAAGAGGCTTCGGAACACACGCAGAAATGCTGCAGGGCGA
AACGCTAGATAGGAATGCCGGGATTTTGCTCGAATTTCTTAATACACAAACCGTAACGGTTTCAG
GATCCAGATCTCATCACCATCACCATCACTAA
NNN: linker and TE domain of SrfC
GGG: CCG in wild type
GrsB pTrc99a
ATGAGTACATTTAAAAAAGAACATGTTCAGGATATGTATCGTTTATCTCCCATGCAGGAAGGCAT
GTTGTTTCACGCATTACTTGATAAAGATAAAAATGCTCACCTGGTACAAATGTCTATCGCGATCG
AAGGTATCGTGGATGTGGAGCTGCTTAGTGAAAGCTTGAACATATTGATTGATAGATACGATGTG
TTTAGAACAACATTCTTACATGAAAAAATTAAACAACCGCTTCAGGTAGTGCTAAAGGAACGGCC
TGTTCAGCTTCAATTTAAAGACATATCATCCTTAGATGAAGAAAAAAGAGAACAGGCTATTGAGC
AGTATAAGTATCAAGATGGGGAAACAGTCTTTGATTTAACAAGAGATCCCTTGATGAGAGTAGCT
ATTTTTCAAACTGGTAAGGTTAACTACCAAATGATCTGGAGCTTCCACCATATTTTAATGGATGG
TTGGTGCTTCAACATTATATTTAATGACTTGTTCAATATATATCTGTCATTAAAAGAGAAGAAAC
CTCTTCAGTTAGAGGCGGTGCAACCATATAAGCAGTTTATTAAGTGGCTTGAAAAACAAGATAAA
CAGGAAGCTCTTCGCTACTGGAAAGAACATTTAATGAATTATGATCAATCAGTAACATTACCTAA
AAAGAAAGCAGCTATTAATAATACTACATATGAACCAGCACAGTTTCGTTTTGCGTTTGACAAAG
TGCTTACCCAGCAGCTGCTTCGTATTGCCAATCAAAGCCAAGTAACACTAAATATTGTTTTTCAA
ACAATATGGGGGATTGTACTTCAAAAATACAATTCCACTAATGATGTTGTATATGGCTCTGTTGT
ATCAGGCCGTCCTTCTGAAATATCGGGAATTGAGAAAATGGTTGGACTATTTATTAATACTCTTC
CATTACGTATCCAAACGCAAAAAGATCAATCATTTATTGAATTAGTAAAGACTGTTCATCAAAAC
GTCCTTTTCTCGCAACAGCATGAGTATTTTCCATTGTATGAAATACAAAATCATACAGAATTAAA
ACAGAATCTGATTGATCATATTATGGTAATTGAAAATTATCCTTTAGTAGAAGAATTGCAAAAGA
ATAGTATCATGCAAAAAGTAGGGTTTACAGTTCGTGATGTCAAAATGTTTGAACCAACTAATTAT
GATATGACAGTTATGGTTTTACCTCGTGATGAAATTAGTGTCCGACTCGATTATAACGCAGCCGT
36
TTATGATATAGATTTCATAAAAAAAATTGAAGGTCACATGAAAGAAGTGGCTTTATGCGTGGCAA
ATAATCCACATGTGTTAGTACAGGACGTTCCTCTGCTTACAAAGCAAGAAAAACAACATTTATTG
GTAGAGCTGCATGATTCGATAACAGAGTATCCTGATAAGACGATTCATCAGTTATTTACAGAACA
GGTAGAAAAAACACCAGAGCATGTGGCAGTTGTATTCGAAGATGAGAAAGTGACCTATAGAGAGC
TGCATGAGAGATCTAATCAATTAGCCAGATTCTTAAGAGAAAAAGGCGTAAAAAAAGAAAGCATC
ATAGGCATTATGATGGAGCGTTCAGTTGAAATGATTGTTGGGATCTTAGGGATTTTAAAAGCTGG
TGGAGCTTTTGTGCCTATTGATCCTGAATATCCAAAAGAAAGAATCGGCTATATGTTAGATTCTG
TACGGCTAGTACTTACACAACGCCATTTAAAGGATAAATTTGCTTTTACGAAAGAAACGATAGTA
ATTGAAGATCCAAGTATTTCACACGAGTTAACTGAAGAAATAGATTATATTAATGAATCAGAGGA
CTTGTTTTATATTATTTATACATCAGGAACAACAGGTAAACCAAAAGGGGTTATGCTAGAGCACA
AAAACATCGTTAATCTGCTTCATTTTACTTTCGAGAAAACAAATATCAACTTTAGTGACAAAGTA
TTACAGTATACAACATGCAGTTTTGACGTGTGTTACCAAGAAATTTTTTCGACGCTCTTGTCTGG
AGGGCAATTATATCTTATTAGGAAAGAAACTCAACGCGATGTAGAGCAATTATTTGATTTAGTAA
AACGTGAAAATATTGAAGTATTATCCTTTCCTGTGGCTTTTCTAAAATTTATTTTCAATGAAAGA
GAATTTATCAATCGTTTTCCAACTTGCGTGAAACATATTATCACAGCAGGAGAACAATTAGTAGT
TAACAATGAGTTTAAACGTTATTTGCATGAACATAACGTACATTTACACAATCATTATGGTCCAT
CAGAAACGCATGTTGTTACCACCTATACTATTAATCCTGAAGCTGAAATTCCTGAATTACCACCG
ATAGGAAAACCTATCTCCAATACATGGATTTATATTTTGGATCAAGAACAACAACTACAACCACA
AGGAATTGTAGGAGAGTTATATATTTCGGGCGCAAATGTTGGAAGAGGATATTTGAATAATCAAG
AATTAACGGCAGAAAAATTCTTTGCAGATCCCTTTAGGCCAAACGAACGGATGTACCGAACAGGG
GATTTAGCAAGGTGGTTGCCAGACGGAAATATCGAATTTTTAGGAAGAGCCGATCATCAGGTGAA
AATTAGGGGGCATCGAATAGAGCTTGGTGAGATCGAGGCACAATTATTAAATTGTAAGGGTGTAA
AAGAAGCTGTTGTTATCGATAAAGCGGATGATAAAGGCGGAAAATATTTATGTGCCTATGTTGTT
ATGGAAGTAGAAGTAAATGACTCTGAGCTTCGAGAATATTTGGGGAAAGCTTTGCCTGATTATAT
GATCCCGTCGTTCTTTGTTCCGTTGGATCAGCTGCCGCTTACACCAAACGGAAAAATAGACAGAA
AATCTCTTCCGAATCTAGAGGGGATTGTGAATACAAACGCAAAATATGTAGTACCTACAAATGAG
CTGGAAGAAAAATTGGCTAAAATCTGGGAAGAAGTACTTGGGATTTCTCAGATCGGTATACAAGA
CAATTTCTTTTCGTTAGGCGGGCATTCTCTTAAAGCCATTACGCTTATTTCCCGTATGAACAAAG
AGTGTAATGTAGACATTCCTCTACGTTTGTTATTTGAAGCACCAACCATTCAGGAAATCTCTAAT
TATATAAACGGGGCAAAGAAAGAAAGCTATGTTGCCATTCAGCCTGTACCAGAACAAGAGTACTA
TCCTGTATCATCAGTTCAAAAAAGAATGTTTATTCTTAATGAATTTGATCGTTCAGGTACGGCCT
ATAATTTACCTGGTGTTATGTTTCTAGATGGAAAATTGAACTACCGACAATTGGAAGCAGCGGTA
AAAAAATTAGTTGAGCGACATGAAGCGCTGCGTACTTCCTTTCATTCAATTAATGGGGAACCAGT
TCAGCGGGTGCATCAAAATGTAGAACTGCAGATTGCTTATTCAGAGTCAACGGAAGATCAGGTGG
AGCGAATTATTGCGGAATTTATGCAACCATTTGCTCTTGAAGTTGCTCCGTTACTTCGTGTAGGT
CTTGTTAAATTGGAGGCAGAACGTCATCTATTTATAATGGATATGCATCATATCATCTCGGATGG
GGTATCCATGCAGATCATGATTCAAGAAATTGCTGATTTGTATAAAGAAAAGGAACTTCCTACGT
TAGGCATTCAATATAAAGACTTTACTGTTTGGCATAATCGCTTGCTTCAATCGGATGTTATTGAA
AAACAAGAAGCTTACTGGCTGAACGTATTTGCAGAAGAGATTCCAGTATTGAATCTACCGACCGA
TTACCCAAGACCAACCATTCAAAGCTTTGATGGTAAAAGATTTACATTCAGTACAGGAAAGCAGC
TTATGGATGATTTATACAAGGTGGCAACAGAAACAGGAACAACACTATATATGGTTTTACTTGCT
GCGTATAATGTTTTCTTATCGAAGTATTCCGGGCAAGATGACATCGTTGTAGGAACACCGATTGC
TGGTAGGTCTCATGCTGATGTGGAAAATATGCTGGGGATGTTTGTAAATACATTAGCAATAAGAA
GTCGTTTAAATAATGAGGATACTTTTAAAGATTTTTTAGCAAATGTAAAACAAACGGCTTTGCAT
GCCTATGAAAATCCAGATTACCCATTTGATACGCTTGTCGAAAAGTTGGGTATACAGAGAGATTT
AAGTAGAAATCCATTATTTGATACGATGTTTGTTTTGCAAAATACGGATAGAAAGTCTTTTGAGG
TTGAACAGATAACGATTACACCATATGTTCCAAATAGCAGACATTCTAAATTTGATCTTACATTA
GAGGTTAGCGAAGAACAAAATGAGATTTTATTATGCCTAGAATATTGCACTAAATTATTTACGGA
TAAAACAGTTGAAAGAATGGCTGGTCATTTTTTACAGATCTTGCATGCAATTGTTGGGAACCCAA
CGATTATAATATCAGAAATCGAGATATTGTCTGAAGAAGAAAAACAACATATTTTATTCGAGTTC
AACGATACGAAAACCACATATCCACATATGCAAACAATTCAAGGATTATTTGAGGAACAGGTGGA
GAAGACGCCCGACCATGTTGCAGTTGGATGGAAAGACCAAACATTAACGTATCGGGAACTTAACG
AAAGAGCGAATCAGGTCGCAAGAGTCTTACGGCAAAAAGGAGTCCAACCCGATAATATCGTGGGA
37
TTGCTGGTTGAGCGTTCACCTGAAATGCTCGTGGGTATCATGGGAATTCTTAAAGCAGGGGGAGC
TTATTTACCTCTTGATCCGGAGTACCCAGCGGATAGAATTTCGTACATGATACAAGATTGTGGTG
TACGCATTATGCTTACCCAACAGCATCTTTTATCTTTAGTACATGATGAATTTGATTGTGTTATT
TTGGATGAAGACAGTTTGTACAAGGGGGATTCTTCCAATTTGGCTCCGGTTAACCAGGCCGGGGA
TTTAGCCTACATCATGTACACTTCTGGTTCTACAGGAAAGCCTAAAGGTGTTATGGTAGAACATC
GAAATGTGATTCGCCTTGTGAAAAATACAAATTATGTTCAGGTCCGCGAAGACGATCGTATAATA
CAGACCGGAGCAATTGGATTCGATGCACTGACATTTGAAGTTTTTGGCTCATTGCTGCATGGAGC
TGAATTGTATCCTGTTACTAAAGACGTGCTATTAGATGCAGAGAAACTACACAAATTTTTACAAG
CGAATCAAATTACGATTATGTGGTTAACTTCTCCGTTATTTAACCAATTGTCACAAGGAACCGAA
GAGATGTTTGCTGGCCTTCGCTCCCTAATTGTAGGTGGAGATGCCTTGTCTCCGAAACACATCAA
TAATGTAAAGCGAAAATGCCCTAATCTGACTATGTGGAACGGTTACGGCCCAACAGAAAACACCA
CTTTTTCTACATGCTTTCTTATTGATAAAGAATATGATGACAATATTCCGATAGGGAAGGCCATT
AGTAATTCAACAGTGTATATCATGGACCGGTATGGCCAGCTTCAGCCGGTGGGTGTACCAGGAGA
ATTATGTGTAGGAGGGGATGGGGTTGCCAGGGGATATATGAATCAGCCTGCATTAACAGAAGAGA
AGTTTGTCCCAAATCCATTCGCTCCTGGTGAGAGAATGTATCGCACGGGGGATTTGGCAAGATGG
TTGCCTGATGGAACAATTGAGTATTTAGGTCGTATTGATCAGCAGGTGAAAATCAGGGGCTACCG
TATTGAACCGGGAGAGATTGAAACGCTTCTTGTGAAGCACAAAAAAGTCAAAGAATCGGTAATCA
TGGTAGTAGAGGATAATAATGGACAAAAGGCTCTATGCGCTTATTACGTTCCGGAAGAAGAAGTA
ACGGTATCTGAACTGAGGGAATATATAGCTAAAGAGTTGCCTGTTTACATGGTTCCAGCCTATTT
TGTACAGATTGAACAAATGCCTCTTACACAGAACGGTAAAGTAAATCGAAGCGCGTTACCAAAAC
CAGATGGTGAATTTGGTACAGCAACCGAATATGTAGCGCCTAGCAGCGACATTGAAATGAAGCTG
GCAGAGATTTGGCATAATGTGTTAGGGGTAAACAAAATCGGGGTACTGGATAACTTCTTTGAATT
AGGTGGTCATTCATTAAGAGCTATGACAATGATTTCCCAGGTACATAAAGAGTTCGACGTTGAAT
TGCCATTAAAAGTGTTATTTGAAACACCAACGATCTCTGCATTAGCTCAATACATTGCTGATGGA
GAAAAAGGAATGTACCTGGCCATTCAACCTGTTACCCCGCAGGATTACTATCCAGTATCATCTGC
GCAAAAGAGGATGTACATCCTTTATGAATTTGAAGGGGCTGGCATTACCTATAATGTACCTAATG
TAATGTTTATAGAAGGAAAGCTGGATTATCAGCGCTTTGAATACGCTATAAAAAGTTTGGTAAAT
CGACATGAGGCGCTTCGAACGTCTTTCTATTCGCTTAATGGAGAACCAGTTCAGCGTGTACATCA
AAATGTAGAGCTACAGATTGCTTATTCGGAGGCGAAAGAAGATGAGATAGAGCAAATTGTAGAAA
GCTTTGTTCAACCATTTGACCTTGAAATAGCTCCGCTGCTTCGCGTAGGGCTTGTTAAATTGGCA
TCGGATCGCTATTTATTCCTAATGGATATGCATCATATTATCTCAGATGGTGTATCAATGCAAAT
TATAACAAAAGAAATTGCCGACTTATATAAAGGAAAAGAGCTTGCTGAACTGCATATTCAGTATA
AAGATTTTGCTGTATGGCAAAACGAATGGTTTCAATCTGACGCTCTTGAAAAACAGAAAACGTAT
TGGTTGAACACCTTTGCAGAGGATATTCCGGTTTTAAATTTGTCAACTGATTATCCAAGACCGAC
AATTCAAAGTTTTGAAGGAGATATTGTCACGTTTAGTGCAGGGAAGCAACTTGCGGAAGAATTGA
AACGCCTGGCTGCAGAAACAGGGACGACTTTGTATATGCTTCTGTTAGCGGCGTACAATGTACTT
TTACACAAATACTCGGGACAGGAAGAAATTGTAGTAGGAACGCCTATTGCCGGGCGATCTCACGC
AGATGTGGAAAATATTGTTGGGATGTTTGTCAATACGCTTGCATTGAAAAATACCCCTATAGCCG
TACGCACCTTCCACGAATTCCTGTTGGAAGTAAAACAAAATGCTTTAGAAGCTTTTGAAAATCAA
GACTATCCATTTGAAAATTTGATAGAGAAGCTGCAAGTGCGTCGCGACTTAAGTCGCAATCCATT
ATTTGATACAATGTTTAGCCTAAGCAATATTGACGAACAAGTAGAGATAGGGATTGAGGGATTGA
ACTTCAGCCCATATGAAATGCAGTATTGGATTGCAAAATTTGATATTTCATTCGATATTTTAGAA
AAGCAAGATGACATTCAATTTTATTTTAACTATTGCACGAATCTGTTTAAAAAAGAAACGATAGA
ACGATTAGCGACACACTTTATGCATATTTTACAGGAGATTGTTATTAATCCTGAGATTAAGTTAT
GTGAAATTAATATGCTGTCCGAAGAAGAACAGCAGCGTGTCCTGTATGACTTTAATGGCACAGAT
GCAACCTACGCTACGAATAAAATATTCCATGAGTTATTTGAAGAACAGGTTGAAAAAACACCAGA
TCATATAGCGGTGATAGATGAAAGAGAAAAGCTTTCCTATCAGGAGCTTAATGCGAAAGCGAATC
AGCTGGCACGAGTGCTGCGCCAAAAAGGAGTACAGCCTAATAGCATGGTAGGTATTATGGTAGAT
CGCTCACTCGACATGATTGTAGGAATGCTTGGGGTTTTAAAAGCAGGAGGAGCATATGTGCCTAT
CGATATAGACTATCCTCAGGAACGGATTAGCTACATGATGGAAGATAGTGGTGCAGCGCTCTTGT
TAACACAACAAAAGTTGACACAGCAAATTGCGTTTTCTGGTGACATTTTGTATCTTGACCAAGAA
GAATGGCTTCATGAGGAAGCTTCAAATTTAGAACCCATCGCTCGTCCGCAGGATATAGCCTATAT
CATTTACACTTCTGGTACAACCGGAAAGCCAAAAGGTGTGATGATTGAGCATCAAAGCTATGTGA
38
ATGTAGCAATGGCATGGAAAGATGCCTATCGGTTAGATACATTCCCGGTCCGTTTGCTTCAGATG
GCTAGCTTTGCCTTTGACGTATCTGCAGGTGATTTTGCCAGAGCACTACTTACAGGTGGGCAATT
AATTGTATGTCCAAATGAAGTAAAGATGGACCCAGCTTCTTTATATGCCATTATTAAGAAATATG
ACATTACTATTTTTGAAGCAACGCCTGCTCTAGTGATTCCATTGATGGAGTATATTTATGAACAG
AAGCTGGATATTAGCCAGTTACAGATTCTGATTGTCGGATCGGACAGTTGTTCGATGGAAGACTT
TAAAACCTTGGTTTCCCGTTTTGGTTCAACTATACGTATTGTGAATAGCTATGGAGTAACCGAAG
CGTGCATTGATTCTAGCTATTATGAACAACCGCTTTCTTCGTTACATGTAACAGGAACTGTACCG
ATTGGAAAACCGTACGCTAACATGAAAATGTATATTATGAATCAATATTTGCAGATTCAGCCTGT
AGGTGTAATTGGAGAATTATGTATTGGAGGAGCCGGGGTTGCCCGTGGATATTTAAATAGACCGG
ACTTAACAGCAGAAAAGTTTGTCCCTAATCCTTTTGTTCCAGGTGAAAAGCTGTATCGAACAGGC
GACTTGGCAAGATGGATGCCGGATGGGAATGTTGAGTTTCTTGGTCGAAATGACCATCAGGTGAA
AATCAGAGGGATTCGAATCGAGCTTGGAGAAATCGAAGCACAACTGCGTAAACATGATAGCATAA
AAGAAGCAACTGTGATCGCAAGAGAAGATCACATGAAAGAGAAATATTTATGTGCGTATATGGTG
ACCGAAGGAGAAGTAAATGTAGCTGAACTGCGTGCGTATCTAGCAAATGATCTGCCTGCGGCAAT
GATTCCGTCATATTTTGTATCGCTCGAAGCAATGCCACTTACTGCTAATGGAAAAATTGATAAGC
GATCTTTACCAGAGCCCGATGGTTCCATATCGATAGGAACAGAATATGTAGCTCCGCGTACCATG
CTTGAGGGAAAACTAGAAGAGATATGGAAAGATGTATTGGGTTTACAGCGTGTTGGCATTCACGA
TGACTTCTTTACAATAGGTGGCCATTCATTGAAGGCTATGGCTGTTATTTCGCAAGTTCATAAAG
AATGCCAGACTGAAGTTCCTCTGCGTGTCTTATTTGAAACACCTACCATTCAAGGACTGGCTAAA
TATATAGAGGAGACGGACACAGAGCAATATATGGCTATTCAGCCGGTTAGCGGACAGGACTATTA
TCCAGTATCATCAGCACAAAAGAGAATGTTTATTGTTAATCAATTTGATGGAGTAGGAATTAGCT
ACAATATGCCTTCCATCATGCTGATTGAAGGAAAACTTGAGCGAACACGCTTGGAATCAGCATTT
AAAAGATTGATAGAACGACATGAGAGCCTTCGAACATCTTTTGAAATAATAAATGGTAAGCCTGT
ACAGAAGATTCATGAGGAAGTTGATTTCAATATGTCCTATCAGGTGGCTTCTAATGAACAAGTAG
AGAAGATGATCGATGAGTTCATTCAGCCTTTCGATTTAAGTGTTGCACCGCTGCTTCGTGTGGAA
CTTTTAAAATTGGAAGAAGACCGTCATGTGCTTATATTTGATATGCATCATATTATCTCAGATGG
TATATCTTCCAATATTTTGATGAAAGAATTAGGAGAACTATATCAAGGTAATGCTTTACCAGAAC
TTCGTATTCAATACAAGGATTTCGCTGTATGGCAAAATGAGTGGTTCCAGTCAGAAGCCTTTAAA
AAGCAAGAAGAATACTGGGTAAATGTCTTCGCAGATGAACGCCCGATTCTGGATATACCGACGGA
TTATCCAAGGCCGATGCAACAAAGCTTTGATGGTGCTCAACTTACATTTGGAACCGGAAAGCAGC
TTATGGATGGGTTATACAGGGTAGCAACGGAAACGGGAACAACGCTTTATATGGTTTTGCTTGCG
GCATATAATGTTCTTCTTTCCAAATATTCTGGTCAAGAAGATATTATTGTAGGGACACCGATTGT
GGGTAGATCCCATACTGACCTTGAGAATATTGTCGGGATGTTTGTCAACACGTTAGCAATGAGAA
ATAAACCGGAAGGAGAAAAGACGTTCAAAGCATTTGTATCAGAAATAAAGCAGAATGCACTAGCG
GCTTTTGAGAATCAGGATTATCCATTTGAAGAGCTTATCGAAAAACTAGAGATACAAAGGGACTT
AAGCAGAAATCCATTATTTGATACGCTCTTTAGCCTTCAAAACATAGGTGAAGAATCATTTGAAC
TAGCCGAATTAACATGCAAACCTTTCGATTTGGTAAGCAAATTAGAGCATGCCAAGTTTGATCTG
AGTCTTGTGGCAGTAGAAAAAGAGGAAGAAATTGCATTTGGGCTTCAATACTGCACAAAACTGTA
TAAGGAAAAAACAGTTGAACAACTGGCTCAACATTTTATTCAAATAGTAAAAGCAATTGTAGAAA
ATCCAGATGTCAAATTATCTGATATTGATATGTTATCTGAAGAAGAGAAGAAACAAATCTTACTT
GAGTTCAATGATACGAAAATACAATATCCGCAGAATCAAACAATACAGGAATTGTTTGAAGAGCA
AGTGAAGAAAACACCTGAACATATAGCAATCGTATGGGAAGGGCAAGCATTAACCTATCATGAGC
TAAATATAAAAGCTAATCAGTTAGCTCGTGTATTACGAGAAAAAGGGGTAACCCCTAATCATCCT
GTAGCGATTATGACGGAACGCTCATTAGAGATGATCGTAGGTATCTTTAGTATTTTGAAAGCAGG
AGGAGCATATGTTCCAATTGATCCAGCCTATCCACAAGAACGTATTCAATACTTGCTTGAAGATA
GCGGAGCGACGCTACTGCTTACTCAGTCACATGTATTAAATAAATTACCGGTCGATATCGAATGG
TTGGATCTTACAGATGAACAAAACTATGTAGAAGATGGTACCAATCTTCCATTTATGAATCAGTC
AACAGATCTTGCCTATATTATTTATACATCCGGTACAACAGGCAAGCCTAAAGGGGTTATGATTG
AACATCAAAGCATCATCAACTGCCTGCAATGGCGGAAGGAAGAATACGAATTTGGACCAGGGGAT
ACGGCTCTACAAGTGTTTTCCTTTGCTTTTGATGGATTTGTAGCAAGTTTGTTTGCTCCGATTCT
TGCAGGTGCAACGTCTGTTCTCCCTAAGGAGGAAGAAGCAAAAGATCCAGTTGCATTGAAAAAAC
TGATCGCATCAGAAGAGATTACACATTACTACGGTGTGCCTAGTTTGTTTAGTGCCATTCTTGAT
GTTTCTTCTAGTAAGGATTTGCAAAATTTACGCTGCGTCACTTTGGGAGGAGAGAAATTACCGGC
39
TCAAATTGTTAAAAAAATCAAAGAAAAAAATAAAGAAATTGAAGTCAACAACGAATATGGGCCTA
CTGAAAATAGTGTAGTAACTACTATTATGCGCGATATACAGGTAGAACAAGAGATTACTATTGGT
CGCCCATTATCTAACGTAGATGTATATATTGTCAATTGTAATCATCAATTACAACCAGTAGGTGT
AGTAGGGGAATTATGTATTGGTGGACAGGGACTTGCAAGAGGATATTTGAATAAACCAGAGCTTA
CAGCAGATAAATTTGTTGTAAATCCATTCGTACCTGGTGAACGTATGTACAAAACCGGTGACCTT
GCAAAATGGCGCTCAGATGGAATGATTGAATATGTGGGGCGTGTTGATGAACAAGTAAAAGTAAG
AGGATATCGGATTGAGCTTGGTGAAATTGAATCAGCTATCCTAGAATACGAAAAAATTAAGGAAG
CGGTAGTTATGGTTTCGGAGCATACTGCATCTGAACAGATGTTATGTGCTTATATTGTAGGGGAA
GAAGATGTACTGACTCTGGACTTAAGAAGCTATCTAGCAAAATTACTACCAAGTTATATGATTCC
AAACTATTTTATCCAATTGGATAGTATTCCGCTTACACCAAACGGTAAAGTGGATCGTAAAGCAT
TGCCTGAACCTCAAACCATTGGCTTAATGGCAAGGGAGTATGTTGCACCAAGGAATGAAATCGAA
GCACAGCTAGTACTCATTTGGCAAGAGGTATTAGGAATAGAACTGATCGGTATTACCGATAATTT
CTTTGAATTAGGAGGGCATTCTTTAAAGGCAACGCTTTTAGTTGCAAAAATTTACGAGTACATGC
AAATAGAGATGCCATTAAATGTTGTGTTTAAACATTCAACTATTATGAAAATAGCGGAATATATT
ACACATCAAGAATCAGAAAATAATGTACATCAGCCTATTTTGGTAAATGTAGAAGCAGATAGAGA
GGCGCTATCTCTTAACGGCGAGAAGCAAAGAAAAAATATAGAGCTACCTATTCTGCTAAACGAAG
AAACAGATCGAAACGTATTCTGCTTCGCGCCCATTGGTGCACAAGGTGTTTTTTATAAAAAGCTT
GCTGAACAAATCCCTACTGCATCCTTGTATGGCTTTGACTTCATTGAAGATGATGATCGAATTCA
GCAATATATTGAATCGATGATTCAAACTCAGTCAGACGGACAATATGTGCTAATTGGTTATTCTT
CAGGAGGGAACCTGGCTTTTGAAGTAGCAAAAGAAATGGAAAGGCAAGGATATAGTGTATCTGAT
TTGGTCTTGTTCGATGTTTACTGGAAGGGAAAAGTCTTCGAGCAAACAAAAGAAGAAGAAGAAGA
AAACATAAAAATAATAATGGAAGAATTAAGGGAAAATCCAGGAATGTTCAATATGACACGAGAGG
ATTTTGAACTGTATTTTGCGAATGAATTTGTGAAACAAAGTTTCACACGGAAAATGCGCAAATAC
ATGAGTTTTTATACGCAGTTAGTTAATTATGGGGAAGTAGAAGCTACAATTCACCTTATACAAGC
AGAATTTGAGGAAGAAAAAATTGACGAAAACGAAAAAGCCGACGAAGAAGAAAAAACATATCTAG
AGGAAAAATGGAATGAAAAAGCATGGAACAAAGCAGCAAAAAGATTTGTAAAATATAACGGATAT
GGCGCTCATTCTAACATGCTAGGAGGTGATGGTTTAGAGAGAAATTCCTCTATCCTTAAACAGAT
ACTACAAGGGACATTTGTAGTAAAAGGATCCAGATCTCATCACCATCACCATCACTAA
TTA: alternative start site ATG in wild type
40
NMR Spectra
41
42
43
44
45
46
47
48
49
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