1
Supporting
Information for
Tryptophan Extends the Life of Cytochrome P450
Raheleh Ravanfar, Yuling Sheng, Harry B. Gray, Jay R. Winkler
Corresponding authors: hbgray@caltech.edu, winklerj@caltech.edu
Email:
hbgray@caltech.edu,
winklerj@caltech.edu
This PDF file includes:
Supporting
text
Figures
S1
to
S5
Tables
S1
to S
3
Legends
for
Dataset
S1
SI
References
Other
supp
orting
materials
for this manuscript include
the following
:
Dataset S1
2
Supporting
Information Text
Materials and Methods
Protein Expression, Isolation, and Purification
.
The cloning vector employed in this study for
cytochrome P450
BM3
was plasmid pET22. Site-
directed mutagenesis was carried out utilizing the
QuikChange Site-
Directed Mutagenesis kit from Qiagen, with primers designed to introduce the
desired mutations, sourced from Invitrogen. The forward primer sequence was 5'
-
ACGCATGAAAAAAATCACAAAAAAGCGCATAAT
-3', and the corresponding reverse primer
sequence was 5'
-ATTATGCGCTTTTTTGTGATTTTTTTCATGCGT
-3'. Experimental samples
were prepared on ice, containing 2 μL of 10X buffer stock, 2 μL of dNTP, 25 ng of the parent
plasmid, 50 ng of the
forward primer, 50 ng of the reverse primer, 0.4 μL of Pfu DNA polymerase,
and milli
-
Q water to reach a total volume of 20 μL. The Polymerase Chain Reaction (PCR)
protocol was executed on a MJ Research PT150 Minicycler, comprising 18 cycles of 30 s at
95°
C, 30 s at 95°C, 60 s at 55°C, and 9 min at 68°C. Methylated DNA (parent plasmid) was
digested by the addition of 1 μL of Dpn1 enzyme and subsequent incubation at 37 °C for one
hour. The PCR mixtures were stored at –
20 °C until required (
1
).
The amino acid sequence for the wild-
type P450
BM3
(holoprotein) is as follows:
SEQ ID NO:1: gi|142798|gb|AAA87602.1| cytochrome P
-
450:NADPH
-P-450 reductase
precursor [
Bacillus megaterium
]
MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEAC
DESRFDKNLSQALKFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQ
LVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQ
RANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDEN
IR
YQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNE
ALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAI
PQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKS
KKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVAT
LDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTY
QKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTL
SLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRN
YEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAM
AAKT
VCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRV
DEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGP
GTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRM
PNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQV
SEADARL
WLQQLEEKGRYAKDVWAGHHHHHH
The nucleotide sequence for WT P450
BM3
(holoprotein) is as follows:
ATGACAATTAAAGAAATGCCTCAGCCAAAAACGTTTGGAGAGCTTAAAAATTTACCGTT
ATTAAACACAGATAAACCGGTTCAAGCTTTGATGAAAATTGCGGATGAATTAGGAGAAATCTT
TAAATTCGAGGCGCCTGGTCGTGTAACGCGCTACTTATCAAGTCAGCGTCTAATTAAAGAAG
CATGCGATGAATCACGCTTTGATAAAAACTTAAGTCAAGCGCTTAAATTTGTACGTGATTTTG
CAGGAGACGGGTTATTTACAAGCTGGACGCATGAAAAAAATTGGAAAAAAGCGCATAATATC
TTACTTCCAAGCTTCAGTCAGCAGGCAATGAAAGGCTATCATGCGATGATGGTCGATATCGC
CGTGCAGCTTGTTCAAAAGTGGGAGCGTCTAAATGCAGATGAGCATATTGAAGTACCGGAA
GACATGACACGTTTAACGCTTGATACAATTGGTCTTTGCGGCTTTAACTATCGCTTTAACAGC
TTTTACCGAGATCAGCC
TCATCCATTTATTACAAGTATGGTCCGTGCACTGGATGAAGCAATG
AACAAGCTGCAGCGAGCAAATCCAGACGACCCAGCTTATGATGAAAACAAGCGCCAGTTTC
AAGAAGATATCAAGGTGATGAACGACCTAGTAGATAAAATTATTGCAGATCGCAAAGCAAGC
GGTGAACAAAGCGATGATTTATTAACGCATATGCTAAACGGAAAAGATCCAGAAACGGGTGA
GCCGCTTGATGACGAGAACATTCGCTATCAAATTATTACATTCTTAATTGCGGGACACGAAAC
AACAAGTGGTCTTTTATCATTTGCGCTGTATTTCTTAGTGAAAAATCCACATGTATTACAAAAA
GCAGCAGAAGAAGCAGCACGAGTTCTAGTAGATCCTGTTCCAAGCTACAAACAAGTCAAACA
GCTTAAATATGTCGGCATGGTCTTAAACGAAGCGCTGCGCTTATGGCCAACTGCTCCTGCGT
3
TTTCCCTATATGCAAAAGAAGATACGGTGCTTGGAGGAGAATATCCTTTAGAAAAAGGCGAC
GAACTAATGGTTCTGATTCCTCAGCTTCACCGTGATAAAACAATTTGGGGAGACGATGTGGA
AGAGTTCCGTCCAGAGCGTTTTGAAAATCCAAGTGCGATTCCGCAGCATGCGTTTAAACCGT
TTGGAAACGGTCAGCGTGCGTGTATCGGTCAGCAGTTCGCTCTTCATGAAGCAACGCTGGT
ACTTGGTAT
GATGCTAAAACACTTTGACTTTGAAGATCATACAAACTACGAGCTCGATATTAA
AGAAACTTTAACGTTAAAACCTGAAGGCTTTGTGGTAAAAGCAAAATCGAAAAAAATTCCGCT
TGGCGGTATTCCTTCACCTAGCACTGAACAGTCTGCTAAAAAAGTACGCAAAAAGGCAGAAA
ACGCTCATAATACGCCGCTGCTTGTGCTATACGGTTCAAATATGGGAACAGCTGAAGGAACG
GCGCGTGATTTAGCA
GATATTGCAATGAGCAAAGGATTTGCACCGCAGGTCGCAACGCTTG
ATTCACACGCCGGAAATCTTCCGCGCGAAGGAGCTGTATTAATTGTAACGGCGTCTTATAAC
GGTCATCCGCCTGATAACGCAAAGCAATTTGTCGACTGGTTAGACCAAGCGTCTGCTGATGA
AGTAAAAGGCGTTCGCTACTCCGTATTTGGATGCGGCGATAAAAACTGGGCTACTACGTATC
AAAAAGTGCCTGCTTTTATCGATG
AAACGCTTGCCGCTAAAGGGGCAGAAAACATCGCTGAC
CGCGGTGAAGCAGATGCAAGCGACGACTTTGAAGGCACATATGAAGAATGGCGTGAACATA
TGTGGAGTGACGTAGCAGCCTACTTTAACCTCGACATTGAAAACAGTGAAGATAATAAATCTA
CTCTTTCACTTCAATTTGTCGACAGCGCCGCGGATATGCCGCTTGCGAAAATGCACGGTGC
GTTTTCAACGAACGTCGTAGCAAGCAAAGAACT
TCAACAGCCAGGCAGTGCACGAAGCACG
CGACATCTTGAAATTGAACTTCCAAAAGAAGCTTCTTATCAAGAAGGAGATCATTTAGGTGTT
ATTCCTCGCAACTATGAAGGAATAGTAAACCGTGTAACAGCAAGGTTCGGCCTAGATGCATC
ACAGCAAATCCGTCTGGAAGCAGAAGAAGAAAAATTAGCTCATTTGCCACTCGCTAAAACAG
TATCCGTAGAAGAGCTTCTGCAATACGTGGAGCTTCAAGAT
CCTGTTACGCGCACGCAGCTT
CGCGCAATGGCTGCTAAAACGGTCTGCCCGCCGCATAAAGTAGAGCTTGAAGCCTTGCTTG
AAAAGCAAGCCTACAAAGAACAAGTGCTGGCAAAACGTTTAACAATGCTTGAACTGCTTGAA
AAATACCCGGCGTGTGAAATGAAATTCAGCGAATTTATCGCCCTTCTGCCAAGCATACGCCC
GCGCTATTACTCGATTTCTTCATCACCTCGTGTCGATGAAAAACAAGCAA
GCATCACGGTCA
GCGTTGTCTCAGGAGAAGCGTGGAGCGGATATGGAGAATATAAAGGAATTGCGTCGAACTA
TCTTGCCGAGCTGCAAGAAGGAGATACGATTACGTGCTTTATTTCCACACCGCAGTCAGAAT
TTACGCTGCCAAAAGACCCTGAAACGCCGCTTATCATGGTCGGACCGGGAACAGGCGTCGC
GCCGTTTAGAGGCTTTGTGCAGGCGCGCAAACAGCTAAAAGAACAAGGACAGTCACTTGGA
GAAGCACATTTATACTTCGGCTGCCGTTCACCTCATGAAGACTATCTGTATCAAGAAGAGCTT
GAAAACGCCCAAAGCGAAGGCATCATTACGCTTCATACCGCTTTTTCTCGCATGCCAAATCA
GCCGAAAACATACGTTCAGCACGTAATGGAACAAGACGGCAAGAAATTGATTGAACTTCTTG
ATCAAGGAGCGCACTTCTATATTTGCGGAGACGGAAGCCAAATGGCACCTGCCGTTGAAGC
AACGCTT
ATGAAAAGCTATGCTGACGTTCACCAAGTGAGTGAAGCAGACGCTCGCTTATGGC
TGCAGCAGCTAGAAGAAAAAGGCCGATACGCAAAAGACGTGTGGGCTGGGCTCGAGCACC
ACCACCACCACCACTGAGATCCGGCTGCTAACAAAGC
The primers for site-
directed mutagenesis:
W96H
-forward: ACGCATGAAAAAAATCACAAAAAAGCGCATAAT
W96H
-reverse: ATTATGCGCTTTTTTGTGATTTTTTTCATGCGT
W90F
-forward: GGGTTATTTACAAGCTTCACGCATGAAAAAAAT
W90F
-reverse: ATTTTTTTCATGCGTGAAGCTTGTAAATAACCC
Y334F
-forward: GCGTTTTCCCTATTTGCAAAAGAAGAT
Y334F
-reverse: ATCTTCTTTTGCAAATAGGGAAAACGC
All sequences were verified by Laragen (Culver City, California).
Transformation, Amplification and Purification of Plasmid DNA
.
For the transformation, 2 μL
of plasmid for the desired mutant were chilled on ice and then mixed with 100 μL of NovaBlue
competent cells for 5 minutes, followed by a 60-
second heat shock at 42°C. After an additional 2
minutes of incubation on ice, 200 μL
of Super Optimal broth with Catabolite repression (S.O.C)
broth were added to the mixture, and the transformation mixture was then incubated for 45
minutes at 37°C with shaking at 250 rpm. Subs
equently, the transformation mixture was plated
on LB/Agar culture plates containing 100 μg/mL ampicillin and incubated overnight at 37°C.
4
To amplify plasmid DNA, either the purified plasmid DNA or the PCR mixture was introduced into
NovaBlue competent cells. Cultures containing 5 mL of Luria Bertani (LB) broth supplemented
with 100 μg/mL ampicillin and a single
E. coli
colony were grown for 16 hours at 37°C with
shaking at 250 rpm. The cells were then pelleted by centrifugation (10 minutes, 13200 rpm), and
the supernatant was discarded. Plasmid DNA was subsequently extracted using a Qiagen
miniprep kit. To confirm the success of mutagenesis,
20 μL samples were sent to Laragen for
sequencing, along with the required sequencing primers (
1
).
Protein Expression and Purification
.
The P450
BM3
protein was expressed and purified with
modification based on the previously reported protocol (
1
). The pET22b(+) plasmid (0.5 μL),
encoding the full length P450
BM3
under the control of the tac promoter, was transformed into
Escherichia coli
BL21(DE3) competent cells (100 μL) and grown for 16 hours at 37°C on a
Lysogeny Broth (LB) plate supplemented with 100 mg/mL ampicillin. A single colony was then
grown in 5 mL of LB media for 6 to 7 hours at 37°C while sh
aking at 250 rpm and subsequently
used to inoculate 100 mL of Terrific Broth supplemented with 100 mg/mL ampicillin (TB
amp
), which
was then grown overnight at 37°C with shaking at 250 rpm. TB
amp
(0.5 L) was inoculated with the
overnight culture (10 mL) and were shaken at 200 rpm at 37°C. Thiamine (0.5 mM) and power
mix (5 mL per 0.5 L culture) were added after 1.5 h of growth at 37°C, and the cultures were
continued to grow up to 4 h until an opt
ical density of 1.2
–1.8 was reached. The cultures were
cooled down to room temperature on ice water bath and the shaker temperature was reduced to
22°C, then the cultures were induced by adding IPTG (0.5 mM), aminolevulinic acid (1 mM) and
extra trace metal mix (500 μL per 0.5 L of culture). The ×1000 trace metal mix
was prepared
using 50 mM FeCl
3
, 20 mM CaCl
2
, 10 mM MnSO
4
, 10 mM ZnSO
4
, 2 mM CoSO
4
, 2 mM CuCl
2
, 2
mM NiCl
2
, 2 mM Na
2
MoO
4
and 2 mM H
3
BO
3
and sterile filtered. The cultures were allowed to
continue for another 20 hours at 22°C and 200 rpm. Cells were harvested by centrifugation (4°C,
15 min, 3000xg), and the cell pellet was stored at
-80°C.
For the purification, the cell pellet was resuspended in Ni
-
NTA buffer A (25 mM Tris HCl, 200 mM
NaCl, 25 mM imidazole, pH 8.2, 0.5 mL/g of pellet) and lysed by sonication (10 minutes at 30
seconds ON/30 seconds OFF pulse mode and 70% power) on the ice bat
h. The lysate was
centrifuged at 27,000xg for 20 min at 4°C to remove cell debris. The theoretical isoelectric point
(pI) for the wild
-
type P450
BM3
is 5.34, calculated using the Expasy ProtParam tool
(
https://web.expasy.org/protparam/
). The collected supernatant was first subjected to a Ni
-
NTA
chromatography step using HisPur
™
Ni
-
NTA Resin (Cat# 88222, Thermo Fisher Scientific). The
enzyme was eluted from the Ni
-
NTA column using 25 mM Tris HCl, 200 mM NaCl, 300 mM
imidazole, pH 8.2. Ni
-
purified protein was buffer exchanged into 20 mM Tris HCl buffer (pH 8.2)
using a 30 kDa molecular weight cut
-off centrifugal filter. The enzyme was then subjected to
HiTrap
TM
Q HP (5 mL, Cytiva) equilibrated and washed with 10 column volume of exchange
buffer and eluted by elution buffer (20 mM Tris, 1 M NaCl, pH 8.2). The protein was buffer
exc
hanged into Tris
–
HCl buffer (0.1 M, pH 8.2) using a 30 kDa molecular weight cut
-off
centrifugal filter. The protein purity and weight were confirmed using LC
-MS. LC
-MS experiments
were performed using a Waters UPLC chromatography system interfaced with a Waters LCT
Premier XE Electrospray Time
-
of-flight mass spectrometer operated in the positive ion mode. The
UPLC column was a 2.1 x 50 mm i.d. BioResolv RP colu
mn from Waters using water with 0.1%
formic acid and acetonitrile with 0.1% formic acid as eluents. For storage, proteins were portioned
into 100 μL aliquots containing 20% glycerol and stored at
-80°C.
Total Turnover Number (TTN) Measurements
.
TTN values were assessed under varying
NADPH concentrations, both in the presence and absence of ascorbate, by quantifying the moles
of yellow
p
-nitrophenolate generated from 12-p-
Nitrophenoxycarboxylic Acid (12
-
p
NCA) per mole
of enzyme until no additional turnover was detected. The 12-
p
NCA substrate was synthesized
following a previously reported procedure (
1
). In brief, 0.1 μM P450
BM3
was allowed to react with
25 μM of the pNCA substrate and 50 μM, 100 μM, and 300 μM NADPH in a total reaction volume
of 10 mL for a duration of 30 minutes. The impact of 100 μM ascorbate on TTN was also
investigated. Subsequently, the enzyme was separated
from the other components of the
reaction mixture using an Amicon Ultra-
15 centrifugal filter with a mass cutoff of 30 kDa (Millipore,
Bedford, MA). Additional substrate, NADPH and ascorbate were then supplied to the enzyme to
5
enable further reaction cycles, after which no significant additional turnover was observed. TTN
was calculated by considering the total micromoles of product formed per micromoles of enzyme.
Kinetics of Oxygen and NADPH Consumption
.
The kinetics of oxygen and NADPH
consumption (Supplementary Figures 1, 2, and 3) were determined by recording the reaction of
0.1 μM P450
BM3
, 50 μM pNCA substrate and 400 μM NADPH in 4700 μL total reaction volume for
a duration of 20 minutes. Oxygen consumption was measured using
an
Ocean Optics
NeoFox
oxygen sensing system with
FOXY oxygen sensor probe
.
The
system uses a fiber optic
fluorescence probe with proprietary oxygen-
sensing thin-
film coating on the tip, designed for
monitoring oxygen partial pressure in aqueous solution. NADPH consumption was monitored on
an Agilent 8453 diode array spectrophotometer at 340 nm.
Tables
S1
and S2 demonstrate the
kinetic results
of oxygen consumption and NADPH consumption, respectively
.
Hydrogen Peroxide (H
2
O
2
) Assay
.
P450
BM3
(0.1 μM) was allowed to react with 100 μM NADPH
and 50 μM pNCA substrate in a total reaction volume of 10 mL for a duration of 30 minutes. The
enzyme was then supplemented with additional substrate and a source of NADPH to enable
more reaction cycles, after which no significant further turnover was observed. Subsequently, the
enzyme was separated from the other components of the reaction mixture using an Amicon Ultra-
15 centrifugal filter with a mass cutoff of 30 kDa
(Millipore, Bedford, MA). H
2
O
2
concentration was
measured by the peroxide assay kit (ab272537, Abcam, Cambridge, UK). This assay kit is
specifically designed to determine peroxide concentrations in samples without the need for any
prior treatment. The method relies on the chromogenic F
e
3+
-xylenol orange reaction, wherein a
purple complex form as a result of the oxidation of Fe
2+
provided in the reagent by peroxides
present in the sample. The intensity of this color, measured within the range of 540-
610 nm,
serves as a precise indicator of peroxide levels in the sample. The assay's detection range spans
from 0.2 μM to 30 μM H
2
O
2
. In brief, a fresh set of standards was prepared and serially diluted
immediately before use. The premix standard was created by combining 5 μL of Standard (3%
H
2
O
2
) with 495 μL of H
2
O, resulting in a 1:100 dilution within a 1.5 mL Eppendorf tube.
Subsequently, 1470 μL of a 30 μM Premix was prepared by mixing 5 μL of H
2
O
2
(1:100 dilution)
with 1465 μL of distilled water. Finally, the standards were diluted in 1.5 mL centrifuge tubes
according to the Table S
3.
The H
2
O
2
detection reagent was prepared for all samples and standards by combining 2 μL of
Reagent A with 200 μL of Reagent B, both of which were provided in the peroxide assay kit.
Subsequently, 200 μL of the detection reagent was added separately to 80 μL of bot
h the
standards and the samples. The reactions were then incubated at room temperature for 30
minutes. After this incubation period, the optical density was measured at the wavelength range
of 540-
610 nm, with the peak at 585 nm. To calculate the sample peroxide content, the optical
density value of standard #8 (H
2
O) was subtracted from the optical density values of the other
standards. These corrected values were then plotted against known H
2
O
2
concentrations to
generate a standard curve (Supplementary Figures 4 and 5). The sample's peroxide content was
subsequently determined by referring to this standard curve.
Solvent Exposure of P450
BM3
Residues
.
Estimates of solvent exposure for P450BM3 residues
were determined using the Biovia Discovery Studio Visualizer program with the x
-ray crystal
structure coordinates from PDB ID 2IJ2. Parameters used in the analysis: 240 grid points per
atom; probe radius 1
.40 Å.
CYP102 Sequence Alignments.
CYP102 amino acid sequences were aligned using the Clustal
Omega multiple sequence alignment program (
https://www.ebi.ac.uk/Tools/msa/clustalo/
). This
list of CYP102 sequences was taken from Parvez et al. (
2
). The results are provided in the
accompanying Supplementary Information
file: CYP102_alignment_sorted.pdf
6
Fig. S1.
Kinetics of oxygen consumption by WT and W96H P450
BM3
. Fitting parameters are set
out in Table S1.
The relative rates of O
2
and NADPH consumption
are used to identify the
uncoupling pathways. The in the oxidase shunt pathway, the NADPH consumption rate (Figures
S2, S3; Table S2) is twice that of the O
2
consumption rate.
7
Fig. S2
.
Kinetics of NADPH consumption by WT and W96H P450
BM3
.
The relative rates of O
2
and
NADPH consumption are used to identify the uncoupling pathways. The in the oxidase shunt
pathway, the NADPH consumption rate is twice that of the O
2
consumption rate
(Figure S
1; Table
S1
).
8
Fig. S
3.
Initial rates of NADPH consumption.
Fitting parameters are set out in Table S2.
The
relative rates of O
2
and NADPH consumption are used to identify the uncoupling pathways. The
in the oxidase shunt pathway, the NADPH consumption rate is twice that of the O
2
consumption
rate
(Figure S
1; Table S1
).
9
Fig. S
4.
Calibration curve for H
2
O
2
assay.
10
Fig. S
5.
H
2
O
2
produced during P450
BM3
turnover in the presence of pNCA and NADPH.
11
Table S1.
Results
of fits to
oxygen
consumption
kinetics.
Fitting curve equations:
M
O2
= A + B
×
exp(
-t/
τ
)
M
O
2
(t=0) = A + B
dM
O
2
/dt = -
(B/
τ
)
×
exp(
-t/
τ
)
dM
O
2
/dt
(t=0) =
-
B/
τ
M
O
2
:
Oxygen concentration (
μ
M)
t:
time (min)
τ
:
Oxygen consumption time constant
(min)
dM
O
2
/dt
(t=0) =
-
B/
τ
: Initial rate of
o
xygen consumption (
μ
M.min
-1
)
M
O
2
(t=0) = A+B
: Initial
o
xygen concentration (
μ
M)
Parameters
Wild
W96H
A
Repeat 1
197.
3
μ
M
226.3
μ
M
Repeat 2
190.
1
μ
M
227.5
μ
M
B
Repeat 1
78.0
μ
M
48.8
μ
M
Repeat 2
82.
8
μ
M
46.6
μ
M
τ
Repeat 1
3.6
min
5.5
min
Repeat 2
4.1
min
5.5 min
Initial rate
Repeat 1
-
21.7
μ
M
.min
-
1
-
8.9
μ
M
.min
-
1
Repeat 2
-
20.2
μ
M
.min
-
1
-
8.5
μ
M
.min
-
1
Average
-
20.9 ± 1.0
μ
M.min
-
1
-
8.7 ± 0.2
μ
M.min
-
1
12
Table S
2.
Results of fits to NADPH
consumption
kinetics.
Fitting curve equations:
M
NADPH
=
At + B
M
NADPH
(t=0) =
B
dM
NADPH
/dt =
A
dM
NADPH
/dt
(t=0) =
A
M
NADPH
:
NADPH
concentration (
μ
M)
t:
time (min)
dM
NADPH
/dt
(t=0) =
A: Initial rate of
NADPH
consumption (
μ
M.min
-1
)
M
NADPH
(t=0) = B
: Initial
NADPH
concentration (
μ
M)
Parameters
Wild
W96H
Initial concentration (B)
Repeat 1
403.4
μ
M
399.8
μ
M
Repeat 2
401.2
μ
M
399.2
μ
M
Repeat 3
399.5
μ
M
401.8
μ
M
Initial rate
(A)
Repeat 1
-
45.3
μ
M
.min
-
1
-
13.2
μ
M
.min
-
1
Repeat 2
-
39.5
μ
M
.min
-
1
-
20.1
μ
M
.min
-
1
Repeat 3
-
32.0
μ
M
.min
-
1
-
17.0
μ
M
.min
-
1
Average
-
39.0 ± 6.6
μ
M.min
-
1
-
16.8 ± 3.4
μ
M.min
-
1
13
Table S
3.
Table of calibration standard for H
2
O
2
production assay.
Standard #
Premix (μL)
H
2
O (μL)
H
2
O
2
(μM)
1
100
0
30
2
80
20
24
3
60
40
18
4
40
60
12
5
30
70
9
6
20
80
6
7
10
90
3
8
0
100
0
14
Dataset
S1 (
CYP102_alignment_sorted.pdf).
Clustal omega alignment of 245 CYP102 amino
acid sequences.
SI
References
Sample References:
1.
Ravanfar R, Sheng Y, Gray HB, & Winkler JR (2023) Tryptophan
-
96 in cytochrome P450
BM3 plays a key role in enzyme survival.
FEBS Lett.
597(1):59
-
64. DOI: 10.1002/1873
-
3468.14514.
2. Parvez M
, et al.
(2016) Molecular evolutionary dynamics of cytochrome P450
monooxygenases across kingdoms: Special focus on mycobacterial P450s.
Scientific
Reports
6(1):33099. DOI: 10.1038/srep33099