In the format provided by the authors and unedited.

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: hilvert@org.chem.ethz.ch

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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51