www.sciencemag.org/content/
358/6360/215/suppl/DC1
Supplementary
Material
s for
Anti
-Markovnikov alkene oxidation by metal
-oxo
–mediated
enzyme catalysis
Stephan C. Hammer
, Grzegorz
Kubik, Ella
Watkins
, Shan
Huang
, Hannah
Minges
,
Frances H.
Arnold*
*Corresponding autho
r. Email: frances@cheme.caltech.edu
Published
13 October 20
17,
Science
358
, 215
(20
17)
DOI:
10.1126/science.
aao1482
This PDF file includes:
Materials and Methods
Figs. S1 to S11
Tables S1 to S3
References
1
This PDF file includes:
I. Materials and methods
2-4
II. General procedures
5-6
III. Supporting figs. S1 to S11
7-17
IV. Supporting tables S1 to S3
18-20
V. NMR spectra from the isotopic labeling experiment
21-22
VI. Preparative scale reactions
23
VII. NMR spectra for preparative scale reactions
24-27
VIII. HPLC standard curves
28-32
IX. HPLC traces for the
anti
-Markovnikov redox hydration
33-37
X. Chiral GC analysis for enantioselective
anti
-Markovnikov redox hydration
38
XI. NMR characterization and spectra of standard compounds
39-52
2
I.
Materials and
m
ethods
(
A
) All chemicals and solvents were purchased from commercial suppliers (Sigma Aldrich,
Alfa Aesar, Fisher Scientific) and used
without additional purification. The following proteins were
purchased: Lysozyme (Sigma
-
Aldrich, product number: L6876), DNase I (GOLDBIO, catalog
number D
-
300
-
5) and alcohol dehy
drogenase (recombinant, from
E.
coli
, Sigma Aldrich, product
number: 49641).
(
B
)
1
H,
13
C and
19
F NMR spectra were recorded on a Varian Inova 300 MHz or 500 MHz,
or Bruker Prodigy 400 MHz instrument, in CDCl
3
and are referenced to the residual solvent peak.
Data for
1
H NMR are reported in the conventional form: chemical shift (
δ
pp
m), multiplicity (d =
doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet), coupling constant (Hz),
integration. Data for
13
C NMR are reported in terms of chemical shift (
δ
ppm), multiplicity (t =
triplet, q = quartet).
(
C
) Analytic
al high
-
performance liquid chromatography (HPLC) was carried out on
an
Agilent 1260 Infinity instrument using
a Poroshell 120 Eclipse column (Agilent, XDB C18, 4.6 x
5 mm, 2.7
μ
m) with H
2
O and acetonitrile as the
mobile phase. All measurements were perform
ed
with an Agilent 1260 Infinity Diode Array Detector and the wavelength at 210
nm was recorded.
HPLC method for screening: Begin with 28% acetonitrile, hold for 0.3
min, linear gradient to 100%
acetonitrile after 1.2
min, hold for 0.3
min, return to 28% a
cetonitrile from 1.5 to 1.6
min., hold for
0.75
min. Flowrate 2.5
mL/min. HPLC method for quantification: Begin with 10% acetonitrile
,
linear gradient to 75% acetonitrile after 7.5
min, linear gradient to 100% acetonitrile after 7.8
min,
hold for 1.0
min,
back to 10% acetonitrile from 8.8 to 8.9
min., keep for 1.4
min. Flowrate
1.5
mL/
min. HPLC method for the quantification of
a
-
methylstyrene and
trans
-
b
-
methylstyrene
conversions: Begin with 20% acetonitrile, linear gradient to 50% acetonitrile after 6.5
mi
n, linear
gradient
to 75% acetonitrile after 7.5
min, linear gradient to 100% acetonitrile after 8.8
min, hold
for 2.0
min, return to 20% acetonitrile from 10.8 to 10.9
min., hold for 1.4
min. Flowrate
1.5
mL/min.
(
D
) Gas chromatography (GC) analyses were
conducted with a Shimadzu GC
-
17A
instrument equipped with a flame ionization detector using an Agilent J&W HP
-
5 column (30
m x
0.32
mm, 0.25
μm film, part number 19091J
-
413) with helium as carrier gas. Injector temperature:
250°C. Split mode with a split r
atio of 5. Detector temperature: 300°C.
Oven temperature: 90
°C
hold 2
min, 6
°C/min to 100
°C, 40
°C/min to 280°C hold 1
min, 9.17
min total.
(
E
) Chiral GC analysis
was conducted with
an Agilent 7820A instrument equi
pped with a
flame ionization detector
using an Agilent Cyclosil
-
B column (30
m x 0.32
mm, 0.25
μm film, part
number 113
-
6632) with helium as carrier gas. Injector temperature: 200°C. Split mode with a split
ratio of 5. Detector temperature: 300°C. Method for the separation of 2
-
phenylpropional
dehyde,
oven temperature: Begin at 90
°C, 0.1
°C/min to 92.8
°C, 15
°C/min to 240
°C, hold 2
min. Method
for the separation of 2
-
phenyl
-
1
-
propanol, oven temperature: Begin at 105
°C, 0.15
°C/min to
109.5
°C, 40
°C/min to 240
°C, hold 2
min.
Method for the
separation of 1
-
phenyl
-
2
-
propanol,
oven temperature
: Begin at 80
°C, hold 2
min, 8
°C/min to 120
°C, 12
°C/min to 170
°C, 15
°C/min
to 240
°C, hold 5
min.
3
(
F
)
P450
LA1
was cloned into a pET22b(+) vector containing a C
-
terminal 6xHis
-
tag. Gene
and amino ac
id sequence of cytochrome P450
LA1
monooxygenase (Uniprot ID: A0P0F6) which
was used as starting point for the directed evolution.
ATG
GAACGCACTGCAAATCCAGCGGACGTTCCGGCTGGTGGTAAATCTTCTGAAGGCAAGGCGGGTACTCCACCGGCT
GCTGAAGCTCAATGCCCTTTCAGCAAAATGGCAGCAGATTTCGACGCTTTCGCAGGCCCATATCAGGCTGATCCGGCA
GAAGCGCTGCGTTGGTCTCGTGACCAGCTGCCGGTTTTCTATTCCCCGAACCTGGGTTACTGGGTGGTTTCTCGTTAC
GATGATATCAAAGCTGTTTTTCGTG
ACAACATCCTGTTCAGCCCGCGTAACGCTCTGGAAAAAATTACTCCGGCAACC
CCGGAAGCGATGGAGGTCCTGAAAGGTTATGGTTACGCAATGAACCGTACCATGGTTAACGAAGACGAACCAGTTCAC
ATGGAACGTCGTCGTGCACTGATGGGCCACTTCCTGCCGGACAATCTGGAAGCTCGTCAGGAGATGGTACGCCGCCTG
ACCCGCGAAAAAATCGATGCATTCATCGATTCCGGTCGCGTGGATCT
GGTGGAAGCCATGCTGTATGAGGTTCCGCTG
AACGTTGCTCTGCACTTCCTGGGCGTTCCGGAGGATGACATTGCCATTCTGAAAAACTTTTCTGTCGCACACAGCGTC
AACACCTGGGGTAAACCGACCGATGAGCAGCAGGTTGCGATCGCACACGACGTTGGTCAGTTCTGGAACTATGCTGGT
AAAATCATCGAAAAAATGCGCAAGGAACCGGACGGTACCGGTTGGATGCACGAAACCATCCGTAAAAAC
GCAGAAATG
CCGGATATTGTTCCGGATTCTTATGTTCACTCCATGATGATGGCGATCATCGTTGCGGCACACGAGACCACCAGCCTG
GCCTCTGCAGGTATGTTTAAAACCCTGCTGACTCACCGTCAGGCTTGGCAGGATATCTGCGAGGACCCGTCCCTGATT
CCGAACGCAGTTGAGGAGTGTCTGCGTTATAGCGGCTCCATCGTGGCATGGCGTCGTCAAGCTACGGCTGCCACCCGT
ATCGGTGGTGTCG
ACATCCCGGAAGGTGCTAAACTGCTGATCGTTCAAGCATCTGGTAATCAGGATGAGCGTCACTTC
GAGGATGGTGACAAATTTGACATCTACCGCGATAACGCGGTGGACCACCTGACCTTTGGCTACGGTTCTCATCAGTGT
ATGGGCAAAAACATTGCCCGTATGGAGATGCGCATCTTTCTGGAAGAAATGACTCGTCGCCTGCCTCACCTGCAACTG
GCGGAACAGGAATTCACTTACCTGTCCAACACCAG
CTTTCGTGGTCCGGATCATGTGTGGGTCGAATGGGATCCGGAA
AAAAACCCTGAACGTGCCGATCCGAGCCTGGCTAACGGCAACCACCGTTTTCCAGTTGGTGCCCCAGCCCGCCGTGAT
ATTGCACGTAAAATTCGCATCAAAACTGTTCGTCGTGAAGCCGACGGCATCCTGGGCCTGACCATTGAGGATGCAAAG
GGTCGTTCTCTGCCACGTTGGTCCGCTGGTGCGCACATCGAAGTGTGTGTTGACGGC
TTCGACCGTAAATATTCCCTG
TGTGGTCGTGCGGACTCCCGTGACTATGATATCGCAGTTCTGCTGGAAGAAGGCGGTCGCGGTGGTAGCCGTCGTATT
CACGAAGTGGCTGCCGAGGGCCTGGAGCTGCGCCTGCGTGGTCCTTCTAACCTGTTCCGCCTGGACGAACAGGCGCGT
TCCTATGTTCTGATTGCGGGTGGCATTGGTATCACCCCGATCCTGGCAATGGCGGACCACCTGAAAGCCCTGGGTCGT
G
ACTACACCATCCACTACTGCGGTCGTTCTCGTCGTTCTATGGCATTCCTGGACCGTCTGCAGGCAGACCATGGCGAG
CGTCTGTCTGTGCATGCTGGCGATGAGAACCGTCACGCCGAGCTGGCTGGTATTGTTGCCTCCCTGCCGGAAGGTGGC
CAGATTTACGCATGTGGTCCGGAACGTATGATCAGCGAGCTGGAAGATCTGACCGCCCGTCTGCCACATGGCACCCTG
CATTTCGAGCACTTCAGCGCTCA
GGAAACTGCCCTGGACCCGTCTAAAGAAAACGCATTCCAGGTAGAACTGAAAGAT
TCCGGTCTGACTCTGGAGGTGGCTGCGAACGTTACCCTGCTGGATGCACTGCTGGCGTCTGGTATCGATATCTCTTGT
GACTGCCGTGAAGGCCTGTGTGGCTCTTGCGAGGTAGAAGTCCTGGAGGGCGAGATCGACCACCGCGACGTGGTACTG
ACTCGCACCGAACGTGCGGAAAACCGTCGCATGATGTCTTGCTGT
TCCCGCTCTGTAAAAGGCGGTAAGCTGAAACTG
GCACTGCTCGAGCACCACCACCACCACCAC
TGA
MERTANPADVPAGGKSSEGKAGTPPAAEAQCPFSKMAADFDAFAGPYQADPAEALRWSRDQLPVFYSPNLGYWVVSRY
DDIKAVFRDNILFSPRNALEKITPATPEAMEVLKGYGYAMNRTMVNEDEPVHMERRRALMGHFLPDNLEARQEMVRRL
TREKIDAFIDSGRVDLVEAMLYEVPLNVALHFL
GVPEDDIAILKNFSVAHSVNTWGKPTDEQQVAIAHDVGQFWNYAG
KIIEKMRKEPDGTGWMHETIRKNAEMPDIVPDSYVHSMMMAIIVAAHETTSLASAGMFKTLLTHRQAWQDICEDPSLI
PNAVEECLRYSGSIVAWRRQATAATRIGGVDIPEGAKLLIVQASGNQDERHFEDGDKFDIYRDNAVDHLTFGYGSHQC
MGKNIARMEMRIFLEEMTRRLPHLQLAEQEFTYLSNTSFRGPDHVWVEWDPEKNP
ERADPSLANGNHRFPVGAPARRD
IARKIRIKTVRREADGILGLTIEDAKGRSLPRWSAGAHIEVCVDGFDRKYSLCGRADSRDYDIAVLLEEGGRGGSRRI
HEVAAEGLELRLRGPSNLFRLDEQARSYVLIAGGIGITPILAMADHLKALGRDYTIHYCGRSRRSMAFLDRLQADHGE
RLSVHAGDENRHAELAGIVASLPEGGQIYACGPERMISELEDLTARLPHGTLHFEHFSAQETALDPSKENAFQVELK
D
SGLTLEVAANVTLLDALLASGIDISCDCREGLCGSCEVEVLEGEIDHRDVVLTRTERAENRRMMSCCSRSVKGGKLKL
ALLEHHHHHH
4
(
G
)
Gene and amino acid sequence of the
anti
-
Markovnikov oxygenase (aMOx).
ATG
GAGCGCACTGCAAATCCAGCGGACGTTCCGGCTGGTGGTAAATCTTCTGAAGGCAAGGCGGGTACTCCACCGGCT
GCTGAAGCTCA
ATGCCCTTTCAGCAAAATGGCAGCAGATTTCGACGCTTTCGCAGGCCCATATCAGGCTGATCCGGCA
GAAGCGCTGCGTTGGTCTCGTGACCAGCTGCCGGTTTTCTATTCCCCGAACCTGGGTTACTGGGTGGTTTCTCGTTAC
GATGATATCAAAGCTGTTTTTCGTGACAACATCCTGTTCAGCCCGCGTAACGCTCTGGAAAAAATCACTCCGCTGACC
CCGGAAGCGATGGAGGTCCTGAAAGGTTATGGT
TACGCACTGAACCATGCCATGATTAACGAAGACGAACCAGTTCAC
ATGGAACGTCGTCGTGCACTGATGGGCCACTTCCTGCCGGACAATCTGGAAGCTCGTCAGGAGATGGTACGCCGCCTG
ACCCGCGAAAAAATCGATGCATTCATCGATTCCGGTCGCGTGGATCTGGTGGAAGCCATGCTGTATGAGGTTCCACTG
AACGTTGCCCTGCACTTCCTGGGCGTTCCGGAGGATGACATTGCCATTCTGAAAA
AGTTTTCTGTCGCACACAGCGTC
AGCACCTGGGGTAAACCGACCGATGAGCAGCAGGTTGCGATCGCACACGACGTTGGTCAGTTCTGGAACTATGCTGGT
AAAATCATCGAAAAAATGCGCAAGGAACCGGACGGTACCGGTTGGATGCACGAAACCATCCGTAAAAACGCAGAAATG
CCGGATATTGTCCCGGATTCTTATGTTCACTCCATGATGATGGCGATCATCGTTGCGGCACACGAGACCACCAGCCT
G
GCCTCTGCAGGTATGTTTAAAACCCTGCTGACTCACCGTCAGGCTTGGCAGGATATCTGCGAGGACCCGTCTCTGATT
CCGAACGCAGTTGAGGAGTGTCTGCGTTATAGCGGCTCCGTTATGGCATGGCGTCGTCAAGCTACGGCTGCCACCCGT
ATCGGTGGTGTCGACATCCCGGAAGGTGCTAAACTGCTGATCGTTCAAGCATCTGGTAATCAGGATGAGCGTCACTTC
GAGGATGGTGACAAATTTGAC
ATCTACCGCGATAACGCGGTGGACCACCTGACCTTTGGCGTGGGTTCTCACCAGTGT
CTGGGCAAAAACATTGCCCGTATGGAGATGCGCATCTTTCTGGAAGAAATGACTCGTCGCCTGCCTCACCTGCAACTG
GCGGGACAGGAATTCACTTACCTGTCCAACACCAGCTTTCGTGGTCCGGATCATGTGTGGGTCGAATGGGATCCGGAA
AAAAACCCTGAACGTGCCGATCCGAGCCTGGCTAACGGCAACC
ACCGTTTTCCAGTTGGTGCCCCAGCCCGCCGTGAT
ATTGCACGTAAAATTCGCATCAAAACTGTTCGTCGTGAAGCCGACGGCATCCTGGGCCTGACCATTGAGGATGCAAAG
GGTCGTTCTCTGCCACGTTGGTCCGCTGGTGCGCACATCGAAGTGTGTGTTGACGGCTTCGACCGTAAATATTCCCTG
TGTGGTCGTGCGGACTCCCGTGACTATGATATCGCAGTTCTGCTGGAAGAAGGCGGTCGCGGTGG
TAGCCGTCGTATT
CACGAAGTGGCTGCCGAGGGCCTGGAGCTGCGCCTGCGTGGTCCTTCTAACCTGTTCCGCCTGGACGAACAGGCGCGT
TCCTATGTTCTGATTGCGGGTGGCATTGGTATCACCCCGATCCTGGCAATGGCGGACCACCTGAAAGCCCTGGGTCGT
GACTACACCATCCACTACTGCGGTCGTTCTCGTCGTTCTATGGCATTCCTGGACCGTCTGCAGGCAGACCATGGCGAG
CGTCTGTCT
GTGCATGCTGGCGATGAGAACCGTCACGCCGAGCTGGCTGGTATTGTTGCCTCCCTGCCGGAAGGTGGC
CAGATTTACGCATGTGGTCCGGAACGTATGATCAGCGAGCTGGAAGATCTGACCGCCCGTCTGCCACATGGCACCCTG
CATTTCGAGCACTTCAGCGCTCAGGAAACTGCCCTGGACCCGTCTAAAGAAAACGCATTCCAGGTAGAACTGAAAGAT
TCCGGTCTGACTCTGGAGGTGGCTGCGAACG
TTACCCTGCTGGATGCACTGCTGGCGTCTGGTATCGATATCTCTTGT
GACTGCCGTGAAGGCCTGTGTGGCTCTTGCGAGGTAGAAGTCCTGGAGGGCGAGATCGACCACCGCGACGTGGTACTG
ACTCGCACCGAACGTGCGGAAAACCGTCGCATGATGTCTTGCTGTTCCCGCTCTGTAAAAGGCGGTAAGCTGAAACTG
GCACTGCTCGAGCACCACCACCACCACCAC
TGA
MERTANPADVPAGGKSSEGKAGTPPAAEAQCPFSKMAADFDAFAGPYQADPAEALRWSRDQLPVFYSPNLGYWVVSRY
DDIKAVFRDNILFSPRNALEKITPLTPEAMEVLKGYGYALNHAMINEDEPVHMERRRALMGHFLPDNLEARQEMVRRL
TREKIDAFIDSGRVDLVEAMLYEVPLNVALHFLGVPEDDIAILKKFSVAHSVSTWGKPTDEQQVAIAHDVGQFWNYAG
KIIEKMRKEPDGTGWMHETIRK
NAEMPDIVPDSYVHSMMMAIIVAAHETTSLASAGMFKTLLTHRQAWQDICEDPSLI
PNAVEECLRYSGSVMAWRRQATAATRIGGVDIPEGAKLLIVQASGNQDERHFEDGDKFDIYRDNAVDHLTFGVGSHQC
LGKNIARMEMRIFLEEMTRRLPHLQLAGQEFTYLSNTSFRGPDHVWVEWDPEKNPERADPSLANGNHRFPVGAPARRD
IARKIRIKTVRREADGILGLTIEDAKGRSLPRWSAGAHIEVCVD
GFDRKYSLCGRADSRDYDIAVLLEEGGRGGSRRI
HEVAAEGLELRLRGPSNLFRLDEQARSYVLIAGGIGITPILAMADHLKALGRDYTIHYCGRSRRSMAFLDRLQADHGE
RLSVHAGDENRHAELAGIVASLPEGGQIYACGPERMISELEDLTARLPHGTLHFEHFSAQETALDPSKENAFQVELKD
SGLTLEVAANVTLLDALLASGIDISCDCREGLCGSCEVEVLEGEIDHRDVVLTRTERAENRRMMSC
CSRSVKGGKLKL
ALLEHHHHHH
5
II. General procedures
(
A
)
Cloning and library creation
. pET22b(+) was used as a cloning and expression vector
for all enzymes and variants described in this study. The
E.
coli
codon optimized P450
LA1
gene
(Uniprot ID: A0P0F6) was
ordered from IDT containing a C
-
terminal 6xHis
-
tag. Random
mutagenesis of P450's heme domain was performed by error
-
prone PCR using
varying
MnCl
2
concentrations. The resulting PCR products were digested with
Dpn
I, purified by agarose
gel
electrophoresis a
nd ligated into the pET22b(+) vector backbone by Gibson assembly
(
45
)
. After a
n
additional DNA purification step the plasmids were transformed
via
electroporation into
E. cloni
strain BL21(DE3).
Site
-
saturation libraries were generated employing the “22c
-
trick” method
(
46
)
.
The PCR products were gel purified, digested with
DpnI
, ligated using the Gibson assembly Mix,
a
nd used to directly transform
E. cloni
strain BL21(DE3).
(
B
)
Expression of P450 libraries in 96
-
well plates
. The expression and screening of P450
variants was performed in 96
-
well plate format. Individual colonies from P450 libraries were
cultivated in 500
μL of
T
errific
B
roth medi
um
(100
μg
mL
-
1
ampicillin final concentration) for
20
h at 37
°C, 250
rpm using humidity control.
Expression cultures were inoculated with 50
μ
L of
preculture into
610
μL of
T
errific
B
roth medi
um
(100
μg
mL
-
1
ampicillin final concentration).
The
cultures were incubated
for 4
h at 37
ºC, 250
rpm
, then
cooled on ice for 10
min before
induction
(0.2
mM IPTG, 0.5
mM aminolevulinic acid, final concentration).
Induced cells were shaken
for
20
h at 25
ºC, 250
rpm. The cells were harvested (4500
́
g, 5
min, 4
ºC) and s
tored at
-
20
°C for
three days.
(
C
)
Screening of
random mutagenesis
libraries using the Purpald assay
.
(
47
)
Buffer
(0.1
M NaH
2
PO
4
, pH
8.0, 0.15
M NaCl, 0.5% glycerol by
weight, 1
.
0
mg/mL lysozyme, 0
.
2
mg/mL
DNase, 300
μL/well) was added to the cell pellet and lysis was performed by incubating 4
h at
4
°C,
150
rpm. The plate was centrifuged (4000
́
g, 4 °C, 10 min) and 100 μL of lysate w
ere
transferred with an automated liqu
id handling workstation to 96
-
well assay plates containing 5
μL
styrene/DMSO stock solution and 100
μL NADH stock solution in buffer (10
mM styrene, 3.3
mM
NADH final concentration). The plates were incubated for 2
h at 20
°C, 200
rpm. Purpald dissolved
in
2
M NaOH (31
mM, 50
μL/well) was added and the plate
was
incubated for 30 min at room
temperature. Aldehyde activity was analyzed by measuring the absorbance at 538
nm.
1500
-
2500
variants were screened per generation.
(
D
)
HPLC screening of
site
-
saturation
libraries
. Buffer (0.1
M NaH
2
PO
4
, pH
8.0, 0.15
M
NaCl, 2% glycerol by weight, 1
.
0
mg/mL lysozyme, 0
.
2
mg/mL DNase, 300
μL/well) was added
to the cell pellet and lysis was performed by incubating 4
h at 4
°C,150
rpm. The plate was
centrifuged (4000
́
g, 4
°C, 10
min) and 150
μL of lysate
were
transferred with an automated liquid
handling workstation to 96
-
well plate
containing 10
μL styrene stock solution (15
mM final
concentration in DMSO) and 250
μL NADPH/ADH stock solution (2
mM NADPH final
concentr
ation, 1
U/mL final conc. for ADH from Sigma Aldrich No. 49461). The plate was
incubated for 1.5
h at 23
°C, 200
rpm. The
reactions were diluted with 600
μ
L acetonitrile and
incubated for 30
min at room temperature. The plate was centrifuged (4000
g, 4
°C,
5
min) and
150
μL of the supernatant
were
transferred to a 96
-
well assay plate. The amount of phenylethanol
and styrene oxide was analyzed using analytical HPLC.
90 variants per site
-
saturation library were
screened.
(
E
)
Large scale
e
xpression of P450 var
iants
.
E. coli
BL21(DE3) cells transformed with
plasmid encoding P450 variants were grown overnight in 5
mL Luria
-
Bertani medium
(100 ug
mL
-
6
1
ampicillin
final concentration)
at
37
ºC, 250
rpm.
Expression cultures were inoculated with 5 mL
of preculture into
500
mL
T
errific
B
roth medi
um
(100
μg mL
-
1
ampicillin final concentration) in a
2
L flask and incubated for 4
h at 37
ºC, 125
rpm. The flask was cooled on ice for 10 min before
expression was
induced (0.2
mM IPTG, 0.5
mM aminolevulinic acid, final concentration).
Induced
cells were shaken for
20
h at 25
ºC, 125
rpm. The cells were harvested (4500
́
g, 5
min, 4
ºC) and
stored at
-
20 °C.
(
F
)
Bioconversion
s
and determination of total turnover numbe
rs
.
Styrene was
converted in an enzyme cascade together employing the P450 variant and alcohol dehydrogenase
to determine the performance of each variant (total turnover number and
anti
-
Markovnikov
selectivity). Because aldehydes are prone to side reaction
in buffered systems, we chose to combine
our P450 with an alcohol dehydrogenase to prevent aldehyde accumulation by direct reduction to
the corresponding alcohol.
E. coli
BL21(DE3) cells were lysed with lysis buffer (0.1
M NaH
2
PO
4
,
pH
8.0, 0.15
M NaCl, 2%
glycerol by weight, 1
.
0
mg/mL lysozyme, 0
.
2
mg/mL DNase) for 4
h on
ice followed by centrifugation (30
min, 4000
́
g, 4
°C). The supernatant was used as lysate in the
bioconversion reactions of styrene and other substrates. The concentration of P450 enzymes
in
lysate was determined from ferrous carbon monoxide binding difference spectra using the
previously reported protocol
(
48
)
.
For bioconversions, 50
μ
L of lysate (approx. 0.3
μ
M P450 final
conc.)
were
mixed with 742
μ
L NADP
+
buffer solution (0.1
M NaH
2
PO
4
, pH
8.0, 0.15
M N
aCl,
2% glycerol by weight, 1
mM NADP
+
final conc.
) containing
10
U ADH (Sigma Aldrich
No.
49461) and 1
% isopropanol for NADPH cofactor regeneration. 8
μ
L substrate stock in DMSO
(5
mM final substrate conc.) were added to start the reaction in 2
mL
screw
-
top glass vials. The
bioconversion was stopped by adding 800
μ
L acetonitrile after incubating 2
h at room temperature
(400
rpm). The sample was incubated for 30
min at room temperature, centrifuged for 5 min.
(14000
́
g, 25
°C) and the supernatant was
analyzed by
analytical HPLC
.
Calibration curves were
determined
for quantitative HPLC analysis using commercially available, authentic standards. The
total turnover numbers (TTN) were calculated
as ratio of
product
and
P450 concentration
s
.
The
anti
-
Markovn
ikov selectivity was calculated by conc. alcohol / (conc. alcohol + conc. epoxide).
The reactio
ns were performed in triplicate
from at least two biological replicates.
For chiral GC
measurements, the reaction was stopped after 2
h by extracting two times w
ith 500
μ
L ethyl acetate,
combined and used for analysis.
(
G
)
P450
LA1
purification
.
E. coli
BL21(DE3) cells were lysed with lysis buffer (3
mL
/
g
cell pellet, 0.1
M NaH
2
PO
4
, pH
8.0, 0.15
M NaCl, 10% glycerol by weight, 10
mM imidazole, 1
.
0
mg/mL lysozyme,
0
.
2 mg/mL DNase) for 4 h on ice followed by centrifugation (35
min, 20,000
́
g,
4
°C). The protein containing a C
-
terminal 6xHis
-
tag was purified by loading the supernatant on a
nickel NTA column (1
mL HisTrap HP, GE Healthcare, Piscataway, NJ) using an AKT
A purifier.
The column was washed with 4 column volumes buffer A (0.1
M NaH
2
PO
4
, pH
8.0, 0.15
M NaCl,
10% glycerol by weight, 10
mM imidazole) and the protein was eluted using a linear gradient from
100% buffer A to 100% buffer B (0.1
M NaH
2
PO
4
, pH
8.0, 0.
15
M NaCl, 10% glycerol by weight,
10
mM imidazole) over 10 column volumes. The combined fractions were dialyzed with 3
L buffer
(0.1
M NaH
2
PO
4
, pH
8.0, 0.15
M NaCl, 10% glycerol by weight) and concentrated. 50
μ
L aliquots
were frozen using liquid nitrogen
and stored and
-
20°C.
7
III. Supporting Figs. S1 to S
1
1
Fig.
S1
Summary of catalytic strategies for direct and sequential
anti
-
Markovnikov oxidation of
alkenes.
Catalytic protocols for
anti
-
Markovnikov oxidation follow three major strategies:
a
modified
Wacker oxidation, a dehydrogenative oxygenation approach
,
and
a
tandem epoxidation
isomerization cascade.
Strictly speaking, the tandem epoxidation
-
isomerization cascade is not a
direct
anti
-
Markovnikov oxidation. This cascade proceeds in a seque
ntial manner via an epoxide
intermediate that is rearranged in a second step. All current protocols
either depend on precious
metals performing with low turnover numbers
(
29
,
49
–
60
)
often combined with substrates bearing
directing groups
(
58
–
60
)
, do not offer enantiocontrol
(
29
,
49
–
63
)
, utilize complex catalysts that
require multistep synthesis
(
56
,
61
)
and/or use stoichiometric amounts of terminal oxidants such
as iodosylbenzene which generate stoichiometric amounts
of waste
(
29
,
49
,
55
,
57
,
59
–
62
)
.
Catalysts that use earth
-
abundant metals
for aerobic direct
anti
-
Markovnikov oxidation
are
unknown
,
and enantioselective
anti
-
Markovnikov oxidations
are out of reach (
Table
S1
)
.
8
Fig.
S2
Selectivity in the metal
-
oxo
-
mediated
anti
-
Markovnikov oxidation of alkenes.
The
anti
-
Markovnikov selectivity in this reaction is most likely a consequence of the relative
energies of the high
-
energy intermediates in the catalytic cycle. The
a
nti
-
Markovnikov reaction
proceeds via the more stabilized secondary carbocation / radical intermed
iates. Markovnikov
selectivity could be achieved via the less
-
stabilized primary carbocation / radical intermediates.
However, Markovnikov oxidation products have not been observed.
9
Fig.
S
3
The
P450
LA1
-
catalyzed
anti
-
Markovnikov oxidation is a direct
oxidation without an
epoxide intermediate.
(
A
) GC chromatogram after conversion of styrene with P450
LA1
. The reaction yields the
corresponding phenylacetaldehyde
2
and styrene
oxide
3
. (
B
) Same reaction as A,
but
in the
presence of
an alcohol dehydrogena
se (ADH). The ADH reduces p
henylacetaldehyde
2
to the
corresponding 2
-
phenylethanol
2
-
Alc
and recycles the cofactor by isopropanol oxidation.
Chromatograms in (
C
) and (
D
) are derived from the conversion of (
R
)
-
and (
S
)
-
styrene oxide.
Neither epoxide
enantiomer
w
as
converted using the P450
LA1
ADH cascade, thus demonstrating
that epoxides are no
t
substrates for P450
LA1
and not intermediates in the catalytic cycle. Thus,
P450
LA1
catalyzes a direct
anti
-
Markovnikov oxidation and not an epoxidation
-
isomeri
zation
sequence as suggested previously
(
18
)
.
10
Fig.
S4
Purpald assay for high
-
throughput colorimetric aldehyde quantification.
(
A
) The Purpald reagent
(
47
)
was used to quantify aldehyde concentration in high
-
throughput.
Purpald forms a cyclic aminal with aldehydes that rapidly get oxidized to the corresponding purple
-
colored tetrazine. (
B
) The UV/VIS spectrum of the tetrazine product shows
a maximum at 538
nm,
which was used as the absorbance wavelength in the assay. (
C
) Tetrazine formation, measured as
absorbance (538
nm), shows linear dependence on the phenylacetaldehyde concentration.
11
Fig.
S5
Predicted protein structure of the P450
LA1
h
eme domain.
The structure of the P450
LA1
heme domain was predicted using I
-
Tasser
(
64
)
, SWISS
-
MODEL
(
65
)
and Phyre2
(
66
)
. The P450
LA1
heme domain has low sequence identity (<
50%) with
structurally characterized P450s. The structure prediction tools generated models based on the
structures of the P450 monooxygenase PikC (PDB ID: 2WHW)
(
67
)
and a P450 from
Streptomyces
peucetius
(PDB ID: 5IT1)
(
68
)
. The mutations identified in the screening of random mutagenesis
libraries are distributed over the heme domain (
T121A, N201K, N209S, Y385H and E418G
).
All
five mutations contribute to the improved activity, but only the T121A mutation, believed to be in
the active site based on these homology models, significantly enhanced selectivity for the
anti
-
Markovnikov product (from 45% to 55%).
These homology models
were used to identify potential
active site amino acids for iterative site
-
saturation mutagenesis
(
21
)
. The following potential active
site amino acids were mutated and screened during the course of laboratory evolution: L97, A103,
Y116, A117, M118, N119, R120, A121, M122, V123I, N124, H206, S207, A275, V278, S3
25,
I326, V327, W329, R330, S428 and F429 (see Table S2 for more details). In addition, the highly
conserved amino acids of the heme binding site (Cys ligand loop) were targeted for site
-
saturation
mutagenesis: F383, G384, Y385 (H385 in P450
LA1
wild type),
G386, S387, H388, Q389, M391,
G392 and K393.
12
Fig.
S6
The
aMOx
-
catalyzed
anti
-
Markovnikov oxidation is a direct oxidation without an
epoxide intermediate.
P450
LA1
wild type did not show any epoxide aldehyde isomerization activity (see Fig.
S3). To
prove that laboratory evolution did not generate isomerization activity, the corresponding (
R
)
-
and
(
S
)
-
epoxides were incubated with aMOx using the standard biotransforma
tion protocol. This
standard protocol includes an alcohol dehydrogenase and cofactor recycling system to reduce the
generated aldehyde to the corresponding alcohol
2
-
alc
. Since neither the (
R
)
-
nor the (
S
)
-
epoxide
enantiomer were converted under these cond
itions, the epoxide is not an intermediate of the
catalytic cycle. (
A
) HPLC chromatogram of a sample (blue) containing phenylethanol
2
-
Alc
and
styrene oxide
3
is compared to the biotransformation of (
R
)
-
3 (red). No aldehyde or alcohol
formation was observe
d. (
B
) HPLC chromatogram of a sample (blue) containing phenylethanol
2
-
Alc
and styrene oxide
3
is compared to the biotransformation of (
S
)
-
3 (red). No aldehyde or alcohol
formation was observed.