Supporting Information
Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2014
Structural, Functional, and Spectroscopic Characterization
of the Substrate Scope of the Novel Nitrating Cytochrome
P450 TxtE
Sheel C. Dodani, Jackson K. B. Cahn, Tillmann Heinisch, Sabine Brinkmann-Chen,
John A. McIntosh, and Frances H. Arnold*
[a]
cbic_201402241_sm_miscellaneous_information.pdf
S1
Supporting Information
Table S1.
List of oligonucleotides used in this study.
Name
Sequence (5’ to 3’)
NdeI_F
CCTGGTGCCGCGCGGCAGCCATATGATGACAGTACCGAGTCCGCTGGCAG
BamHI_R
GTCGACGGAGCTCGAATTCGGATCCTCAGCGCAGACTCAGTGGG AGGCTT
Figure
partial-
o
using C
S1.
Fo-Fc
o
ccupancy
a
oot.
omit map
o
a
xial water
,
o
f the Txt
E
,
and solve
n
S2
E
active site
n
t tunnel.
M
, showing
c
M
ap is cont
o
c
lear densi
t
o
ured to 3.
5
t
y for the t
r
5
σ
. Image
r
yptophan,
generated
S3
Figure S2.
UV-visible spectra (at 400 nm and 230 nm) and LC-MS chromatograms for the TxtE
nitration reaction with substrate 1. A)
DL
-Tryptophan with purified protein, B)
DL
-tryptophan
with cell lysate and C)
L
-tryptophan in cell lysate.
S4
Figure S2 continued.
UV-visible spectra (at 400 nm an
d 230 nm) and LC-MS chromatograms
for the TxtE nitration reaction with substrate 1. A)
DL
-Tryptophan with purified protein, B)
DL
-
tryptophan with cell lysate and C)
L
-tryptophan in cell lysate.
S5
Figure S3.
UV-visible spectra (at 400 nm and 230 nm) and LC-MS chromatograms for the TxtE
nitration reaction with substrate
4
.
S6
Figure S4.
UV-visible spectra (at 400 nm and 230 nm) and LC-MS chromatograms for the TxtE
nitration reaction with substrate
6
.
S7
Figure S5.
UV-visible spectra (at 400 nm and 230 nm) and LC-MS and extract chromatograms
for the TxtE nitration reaction with substrate
7
.
S8
Figure S6.
UV-visible spectra (at 400 nm and 230 nm) and LC-MS chromatograms for the TxtE
nitration reaction with substrate
8
.
S9
Figure S7.
UV-visible spectra (at 400 nm and 230 nm
) and LC-MS chromatograms for the TxtE
nitration reaction with substrate
9
.
S10
Figure S8.
UV-visible spectra (at 400 nm and 230 nm
) and LC-MS chromatograms for the TxtE
nitration reaction with substrate
10
.
S11
Figure S9.
UV-visible spectra (at 400 nm and 230 nm
) and LC-MS chromatograms for the TxtE
nitration reaction with substrate
11
.
S12
Figure S10.
UV-visible spectra (at 400 nm and 230 nm) and LC-MS chromatograms for the
TxtE nitration reaction with substrate
24
.
S13
Figure S11.
UV-visible spectra (at 400 nm and 230 nm) and LC-MS chromatograms for the
TxtE nitration reaction with substrate
27
.
S14
Figure S12
.
UV-visible spectra (at 400 nm and 230 nm) and LC-MS chromatograms for the
TxtE nitration reaction with substrate
29
.
S15
Figure S13.
Differential UV-visible spectra
of TxtE with derivatives of
L
-tryptophan with
blocked heteroatoms. Substrate binding (250–500 μ
M
) was determined in
E. coli
cell lysates
expressing TxtE (1.6–2.3 μ
M
) in 25 m
M
Tris buffer (pH 8). The substrate number is noted in the
upper left corner of each spectrum. For compound
s that have low signal, a magnified differential
spectrum is shown as an inset in the upper right corner.
S16
Figure S14.
Differential UV-visible spectra of TxtE with
L
-tryptophan derivatives with modified
side chains. Substrate binding (250–500 μ
M
) was determined in
E. coli
cell lysates expressing
S17
TxtE (1.6–2.3 μ
M
) in 25 m
M
Tris buffer (pH 8). The substrate number is noted in the upper left
corner of each spectrum. For compounds that have low signal, a magnified differential spectrum
is shown as an inset in the upper right corner.
S18
Figure S15.
Differential UV-visible spectra of TxtE with
L
-tryptophan derivatives with modified
amino acid motifs. Substrate binding (250–500 μ
M
) was determined in
E. coli
cell lysates
expressing TxtE (1.6–2.3 μ
M
) in 25 m
M
Tris buffer (pH 8). The substrate number is noted in the
upper left corner of each spectrum. For compounds that have low signal, a magnified differential
spectrum is shown as an inset in the upper right corner.
S19
Figure S16.
Analysis of substrate binding to TxtE
by UV-visible spectroscopy. For each
substrate, differential UV-visible spectra are plotted (left) from the titration of TxtE (5 μ
M
) with