S
1
Supporting Information
© Wiley
-
VCH 2016
69451 Weinheim, Germany
Enantioselective Aminohydroxylation of
Styrenyl
Olefins Catalyzed by an
Engineered Hemoprotein
Inha
Cho, Christopher K. Prier, Zhi
-
Jun Jia, Ruijie K. Zhang, Tamá
s Görbe, and Frances H. Arnold
*
Division of Chemistry and Chemical Engineering, California Institute of Technology,
1200 East California Boulevard, Pasadena, California 91125
Email: frances@ch
eme.caltech.edu
DOI: 10.1002/anie.2018XXXXX
Table of Contents
I
.
Experimental Procedures
................................
................................
................................
.........................
...S
2
II.
Supplementary Figures a
nd Tables
................................
................................
................................
.........
...S
7
III.
Synthesis of Substrates and Product Standards
................................
................................
.......................
S
14
IV.
C
haracterization of Pr
oducts and HPLC Calibration
Curv
es
................................
................................
..
S
16
V.
Derivatization of Amino Alcohol Products
................................
................................
.............................
S
20
VI.
Determination of Enantioselectivity and Assignment of Absolute Stereochemistry
..............................
S
21
VII.
Preparative Scale Biocatalytic Reactions
................................
................................
................................
S
32
VIII.
Mechanistic Studies
................................
................................
................................
................................
.
S
34
IX.
Sequence Information
................................
................................
................................
..............................
S
39
X
.
Supplementary References
................................
................................
................................
......................
S
41
S
2
I.
Experimental P
rocedures
a.
General.
Unless otherwise noted, all chemicals and reagents were obtained from su
ppliers Millipore Sigma and
VWR
and were used without further purification. NMR spectra were obtaine
d using a Varian
Innova
300 MHz
, Bruker Prodigy 400 MHz, or Varian Innova
500 MHz instrument, using DMSO
-
d
6
as the
solvent and are referenced to residual solvent signals.
Data for 1H NMR are reported in the
conventional form: chemical shift (δ ppm), multiplicity (s = singl
et, d = doublet, t = triplet, q =
quartet, hept = heptet, m = multiplet, br = broad), coupling constant (Hz), and integration.
Sonication
was performed using a Qsonica Q500 sonicator.
Chemical reactions were monitored using thin layer
chromatography (Merck
60 silica gel plates) and a UV lamp for visualization, if possible.
b.
Chromatography.
Reverse
-
phase high
-
performance liquid chromatography (HPLC) and liquid chromatography
-
mass
spectroscopy (LC
-
MS) were carried out using Agilent 1200 series instruments and
an Agilent XDB
-
C8 column (4.6 x 150 mm, 5 μm) with water and acetonitrile as the mobile phase. Both water and
acetonitrile contained 0.1% acetic acid. Semi
-
preparative HPLC was performed using an Agilent
XDB
-
C18 column (9.4 x 250 mm, 5 μ m) with water and
acetonitrile containing 0.1% acetic acid.
Analytical chiral HPLC was performed using
a Daicel Chiralpak IC column (4.6 x 2
50 mm, 5 μ m)
with hexanes and isopropanol as the mobile phase.
c.
C
l
oning and site
-
saturation mutagenesis
.
Plasmid pET22b(+)
(Novagen)
was used as a cloning and expression vector for all
variants
described
in this
paper
. Site
-
saturation mutagenesis was performed using modified QuikChange
TM
mutagenesis
protocol
using 22
-
codon trick
.
1
The PCR products were further digested with
Dpn
I, purifi
ed with
New England Biolabs gel purification kit, and the gaps were repaired using Gibson Mix
TM
.
2
Without
further purification, 1 μL of the ligation mixture was used to transform
50 μL of
electrocompetent
E.
coli
strain BL21 E. cloni (Lucigen).
d.
Cloning fo
r cytochrome
c
.
The gene encoding
Rhodothermus marinus
cytochrome
c
(UNIPROT ID B3FQS5) was originally
obtained as a single gBlock (IDT), codon
-
optimized for
E. coli
, and cloned using Gibson assembly
into pET22b(+) between restriction sites
Nde
I and
Xho
I
in frame with an N
-
terminal pelB leader
sequence and a C
-
terminal His
-
tag. This plasmid was used as the basis of all
Rma
cytochrome
c
S
3
variants described in this study. All plasmids and ligation mixtures containing the gene encoding a
cytochrome
c
variant
were
co
-
transformed with the cytochrome
c
maturation plasmid pEC86
3
encoding the gene ccmABCDEFG for maturation into BL21 E. cloni (Lucigen).
Cytochrome
c
site
-
saturation
mutagenesis was performed using a further modified protocol
with
22
-
codon trick
. Instead of one long
-
range PCR of the entire plasmid, two PCRs were performed,
pairing the forward site
-
saturation primer mixture with a pET22b(+) internal reverse primer, and the
reverse site
-
saturation primer mixture with a pET22b(
-
) internal forward pr
imer. The two PCR
products were purified together using New England Biolabs gel purification kit, and the gaps were
repaired using Gibson Mix. The ligation mixture was then used directly for co
-
transformation with
the pEC86 plasmid into BL21 E.
c
loni elect
rocompetent cells.
e.
Expression of P411
BM3
,
globin
, and cytochrome
c
variants.
Escherichia coli
BL21 E. cloni
electrocompetent
ce
lls were transformed with pET22
b
(
+
)
constructs
encoding various P411
BM3
and protoglobin
variants. The cells were grown overnight in 6
-
mL Luria
-
Bertani (LB
amp
) medium, supplemented with ampicillin, and 5 mL of this preculture were used to
inoculate 45 mL of Hyperbroth (HB
amp
) medium. The expression culture was incubated at 37 °C and
250 rpm f
or two hours. Then, the expression culture was cooled on ice for 20 minutes and was
induced with 1 mM 5
-
amino
l
evulinic acid (ALA) and 0.5 mM isopropyl β
-
D
-
1
-
thiogalactopyranoside
(IPTG), final concentrations. Cells were allowed to express at 22 °C and 130
rpm for 24 hours. Once
expression was finished, the cultures were centrifuged (4,000 g, 5 minutes, and 4 °C) and the pellets
were resuspended to an OD
600
of 30 in M9
-
N minimal medium at pH 7.4.
To express cytochrome
c
variants,
E. coli
BL21 E. cloni
electr
ocompetent
cell
s were transformed
with pET22b(
+
)
constructs encoding various cytochrome c variants, along with the pEC86 construct
for cytochrome c maturation. The cells were grown overnight in 5 mL Luria
-
Bertani (LB
amp+chl
)
medium, supplemented with ampic
illin and chloramphenicol, and 0.5 mL of this preculture were used
to inoculate 30 mL of Hyperbroth (HB
amp+chl
) medium. The expression culture was incubated at 37 °C
and 250 rpm until an OD600 of 0.5
–
0.7 was reached. Then, the expression culture was cool
ed on ice
for 25 minutes and was induced with 200 μM ALA and 20 μM IPTG, final concentrations. Cells were
allo
wed to express at 2
2
°C and 250 rpm for 20
–
24 hours. Once expression was finished, the
cultures were centrifuged (4,000 g, 5 minutes, 4 °C) and
the pellets were resuspended to an OD
600
of
30 in M9
-
N minimal medium at pH 7.4.
S
4
f.
Purification of cytochrome
c
variants.
Harvested cells from 300
–
600 mL of expression cultures were resuspended in buffer containing 200
mM potassium phosphate, 100 mM
NaCl, and 20 mM imidazole (pH is adjusted to 7.5 at 25 °C).
Cells were lysed by sonication (4 minutes, 1 seconds on, 1 seconds off, 40% intensity; Qsonica Q500
sonicator). The sonication was repeated once more, and then the partially lysed cells were place
d in a
55 °C heat bath for 20 minutes and then moved to 75 °C heat bath for an additional 10 minutes. The
cell debris was removed by two serial centrifugation steps (5,500 g, 20 minutes, 4 °C and then 30,000
g, 30 minutes, 4 °C). The supernatant was filter
ed through a 0.2
-
μ m cellulose acetate filter and
purified using a 1
-
mL Ni
-
NTA column using AKTA purifier FPLC system by running a gradient
from 20 mM to 500 mM imidazole over 10 column volumes. Pure fractions were determined using
SDS
-
PAGE and were pooled
and concentrated using a 3
-
kDA molecular weight cut
-
off centrifugal
filter. The concentrated fractions were washed twice with 0.1 M phosphate buffer at pH 7.5 and were
dialyzed overnight into 0.05 mM phosphate buffer at pH 7.5 using a 3
-
kDA molecular weigh
t cut
-
off
dialysis tubing. The dialyzed protein was frozen on dry ice and stored at
-
20 °C.
g.
Reaction setup and product characterization.
In preparation f
or the reaction, a 250
-
mM glucose solution was prepared in M9
-
N medium (pH 7.4),
filtered through a 0.
2
-
μ m cellulose acetate filter, and placed in the anaerobic chamber for at least a
day. The cells were degassed by sparging with argon for 30 minutes at 4 °C. To 2
-
mL screw cap vials
were added 20 μL of a stock solution containing 1,000 U/mL glucose oxidase
and 14,000 U/mL
catalase prepared in double
-
distilled water. Resuspended cells were added to the screw cap vials, and
all vials containing cells or reagent were immediately transferred into an anaerobic chamber with
oxygen concentration below 15 ppm. In t
he chamber, 40 μL of the glucose solution and the reagents
(typically 200 mM
-
1 M stocks in DMSO) were added. The final reaction volume was 400 μL for all
reactions
on analytical
scale.
The reaction mixture was typically shaken at room temperature for 24
ho
ur at 600 rpm.
After completion of the reaction, a
cetonitrile was added to quench the reaction and
precipitate protein. The mixture was frozen for 4 hours at
-
20 °C and then defrosted at room
temperature for an hour to encourage cell lysis. The defrosted m
ixture was transferred to a 1.7
-
mL
microcentrifuge tube and was centrifuged at 14,000 g for 5 minutes. To determine the yield, 200 μL
of the supernatant were transferred to an HPLC vial with insert and analyzed by reverse
-
phase HPLC
-
MS. Protein concentrati
on in the cell was determined by performing hemochrome assay on the cell
lysate. Lysate was obtained by sonication (4 minutes, 1 seconds on, 1 seconds off, 40% intensity)
followed by centrifugation (4,500 g, 10 minutes, 4 °C) to remove cell debris.
S
5
In orde
r to determine the enantioselectivity
,
acetonitrile was removed from the remaining
supernatant,
and
the solution was
made more basic
by adding 80 μL of 1 M NaOH (aq). For
derivatization, 10
–
15 μL of 10% benzoyl chloride solution in acetonitrile were adde
d, and the
solution was allowed to shake at room temperature (25 °C) and 600 rpm for 4 hours. Derivatized
product was extracted with ethyl acetate and analyzed by chiral normal
-
phase HPLC to determine the
enantiomeric excess (ee).
h.
Reaction screening in 96
-
well plate format.
After
a single
-
site
-
saturation library was generated
, 88 single colonies were
randomly picked and
cultured in 300 μ
L
of
LB medium with 0.1 mg/mL ampicillin (LB
amp
) in a sterilized 96
-
well culture
plate. The plate contained four wells inoculated with parent single colonies, and four sterile wells.
The cells in LB medium were cultured at 37°C, 230 rpm, and 80% relative humidity for 10
-
12 hours.
A separate sterilized
96
-
well culture plate was filled with 1000 μL of Hyperbroth medium containing
0.1 mg/mL ampicillin (HB
amp
) in each well. The new plate with HB
amp
was inoculated with the LB
preculture (50 μL/well)
and incubated at 37°C, 230 rpm, and 80% relative humidity f
or 3 hours. The
plate was cooled on ice for 30 minutes, induced with 20 μM IPTG and 200 μM 5
-
aminolevulinic acid
(final concentrations), and the
n expression was conducted at 2
2
°C and 230 rpm for 20 hours. The
cells were pelleted (4,500 g, 5 minutes), an o
xygen depletion system
4
(20 μL/well of a stock solution
containing 14,000 U/mL catalase from
Corynebacterium glutamicum
and 1,000 U/mL glucose
oxidase from
Aspergillus niger
in M9
-
N medium) was added, and the mixture was gently
resuspended. The 96
-
well pla
te was transferred to
an
anaerobic chamber, where argon
-
sparged 50
mM
D
-
glucose
in 0.1 M potassium phosphate buffer (pH 8.0) was added (300 μL/well). The nitrene
source (10 μL/well, 400 mM in DMSO) and the alkene (10 μL/well, 400 mM in DMSO) were
subsequen
tly added, and the plate was sealed with an aluminum foil
and
shaken at 60
0
rpm at room
temperature for 20
-
24 hours.
Once the plate was taken out of the anaerobic chamber and the seal was removed, acetonitrile
(800 μL/well) and internal standard (10 μL
/well of a 200mM DMSO stock) were added. The wells
were mixed by pipetting and the plate was left to shake at 6
0
0 rpm at room temperature for an
hour
.
The plate was then centrifuged (5,000 g, 5 minutes), and the supernatant (200 μL/well) was filtered
throu
gh an AcroPrep 96
-
well filter plate (0.2 μm) into a shallow 96
-
well plate for reverse
-
phase
HPLC
-
MS
analysis.
S
6
i.
Protein lysate preparation.
Protein
-
containing lysate was prepared from pelleted and resuspended cells adjusted to
a given
OD
600
.
Protein lysate
s
for P450s were obtained by sonication (1 second on, 1 second off, 3 minutes
with 30%
-
40% amplitude, on wet ice
)
in the presence of
B
ugB
uster
TM
reagent. The sonicated cell mixture was
distributed into 2.0
-
mL Eppendorf tubes and then centrifuged (14,000 g, 10 minutes, 4 °C) to remove
cell debris. The supernatant was immediately transferred to a different container, filtered through a
0.
45
μm cellulose filter, and used to determine protein concentration.
Prot
ein lysates for cytochrome
c
variants were obtained by
freezing at
-
20 °C for 3 hours, then thawing and performing
a heat
treatment (55 °C, 25 minutes). The heat
-
treated mixture was centrifuged (5,000 g, 10 minutes, 4 °C)
to remove cell debris. The superna
tant was filtered through a 0.2 μ m cellulose filter.
j.
Determining protein concentration
.
All hemoprotein c
oncentrations were determined in triplicate using the hemochrome assay. A solution
of 1 M NaOH (0.4 mL) was mixed w
ith pyridine (1 mL) in a 1.7 mL
-
Eppendorf tube. The tube was
centrifuged (14,000 g, 1 minutes) to separate the excess aqueous layer and give a pyridine
-
NaOH
solution on top. Separately, a solution of sodium dithionite (10 mg/mL) was prepared in a potassium
phosphate buffer (pH 8.0). To a
cuvette containing 800 μL protein solution in potassium phosphate
buffer (pH 8.0), 50 μL of dithionite solution and 150 μL pyridine
-
NaOH solution were added. The
solution was mixed, the cuvette was sealed with Parafilm, and the UV
-
Vis spectrum
of the redu
ced
hemoprotein was recorded immediately. Hemo
p
rotein concentrations were determined using
ε
550
-
535
=
22.1 mM
-
1
cm
-
1
for heme
c
.
5
This value was also compared with an alternative method of hemochrome
assay where potassium ferricyanide solution is used
,
6
and
the agreement
was within 3
%.
S
7
II.
Supplementary Figures and Tables
Supplementary Table
1
.
Aminohydroxylation of 4
-
vinylanisole
(
3
)
with variants of cytochrome P450
BM3
.
(a)
Variant
Mutations relative to the wild
-
type
P450
BM3
Yield
[%]
pET22b(+) vector
N/A: not expressing any hemoprotein
0.9
P450
BM3
none
0.8
P411
BM3
C400S
4.1
(b)
P411
BM3
T268A
T268A, C400S
2.4
P411
BM3
F87A T268A
(c)
F87A, T268A, C400S
1.5
P411
BM3
F87V T268A
F87V, T268A, C400S
1.2
P411
BM3
CIS T438S
(“P”)
V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,
T268A, A290V, L353V, I366V, C400S, T438S, E442K
1.9
P411
BM3
CIS T438S
A268T
V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,
A290V, L353V, I366V, C400S, T438S, E442K
1.7
“P
-
I263F”
(c)
V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,
T268A, A290V, L353V, I366V, C400S, T438S, E442K
2.4
“
P
-
I263F
”
F263M
P
-
I2
63F I
263M
1.3
“
P
-
I263F
”
F263Y
P
-
I2
63F I
263Y
1.2
“
P
-
I263F
”
A328V
P
-
I263F A328V
3.4
(b)
“P
-
I263F” V87A
A328V
P
-
I263F
F
87A A328V
1.4
“A10”
(d)
P
-
I263F A328V, L437V
1.9
“A10” C400H
P
-
I263F A328V, C400H, L437V
1.2
“A10” C400A
P
-
I263F A328V, C400A, L437V
1.2
“A10” C400
V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,
T268A, A290V, A328V, L353V,
I366V, L437V, T438S, E442K
1.0
P
-
I263F V87A A328V
A268G A82I (“A82I”)
P
-
I263F A82I,
F
87A,
T
268G, A328V
1.1
P
-
I263F V87A A328V
P
-
I263F A82L
, F87A, T
268G, A328V
1.2
S
8
A268G A82L (“A82L”)
"A82L" A78V F263L
E267D
P
-
I263F A78V, A82L,
F
87A, F263L, E267D,
T
268G,
A328V
1.7
“A82L” A78V F263L
P
-
I263F A78V,
A82L, F
87A, F263L,
T
268G, A328V
1.1
P411
BM3
H2
-
4
-
D4
L75A, V78A, F87V, P142S, T175I, M177A, L181A, A184V, S226R,
H236Q, E252G, T268A, A290V, L353V, I366V,
C
400S, L437A,
E442K
4.4
(b)
P411
BM3
H2
-
A
-
10
L75A, V78A,
F87V, P142S, T175I, L181A, A184V, S226R, H236Q,
E252G, T268A, A290V, L353V, I366V, C400S, E442K
3.8
(b)
P411
BM3
H2
-
5
-
F10
L75A, V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,
I263A, T268A, A290V, L353V, I366V, C400S, L437A, E442K
1.5
P411
BM3
CIS I263W
C400H
V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,
I263W, T268A, A290V, L353V, I366V, C400H, E442K
1.1
P411
BM3
CIS L437F
T438Q
V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,
T268A, A290V, L353V, I366V, C400S, L437F, T438Q, E442
K
1.4
P411
BM3
CIS L437F
T438Q L75Y
L75Y, V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,
T268A, A290V, L353V, I366V, C400S, L437F, T438Q, E442K
1.3
P411
BM3
CIS L437F
T438Q L181V
V78A, F87V, P142S, T175I, L181V, A184V, S226R, H236Q,
E252G, T268A,
A290V, L353V, I366V, C400S, L437F, T438Q,
E442K
1.5
P411
BM3
CIS L437F
T438Q L75Y L181I
L75Y, V78A, F87V, P142S, T175I, L181I, A184V, S226R, H236Q,
E252G, T268A, A290V, L353V, I366V, C400S, L437F, T438Q,
E442K
1.3
P411
BM3
CIS I263G
L437F
V78A, F87V, P142S
, T175I, A184V, S226R, H236Q, E252G,
I263G, T268A, A290V, L353V, I366V, C400S, L437F, E442K
1.4
P411
BM3
CIS I263G
L437F V87L
V78A, F87L, P142S, T175I, A184V, S226R, H236Q, E252G, I263G,
T268A, A290V, L353V, I366V, C400S, L437F, E442K
2.0
P411
BM3
CIS I263
G
L437F V87L L181R
V78A, F87L, P142S, T175I, L181R, A184V, S226R, H236Q,
E252G, I263G, T268A, A290V, L353V, I366V, C400S, L437F,
E442K
2.1
P411
BM3
CIS V87T
T438C
V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,
T268A, A290V, L353V, I366V, C400S,
T438C, E442K
1.2
P411
BM3
CIS V87T
I263G L437F T438C
Q674STOP
V78A, F87T, P142S, T175I, A184V, S226R, H236Q, E252G, I263G,
T268A, A290V, L353V, I366V, C400S, L437F, T438C, E442K,
Q674STOP
1.6
(a)
Reactions
were
performed
using
whole cells at OD
600
= 30 with 5 mM of each substrate; results are the
average of duplicate reactions. N.D. = none detected.
S
9
(b) Enantiomeric excess was measured for P450
BM3
variants that gave
highest yields
. The ee ranged from 0 to
11%, with P411
BM3
giving 11%
ee
.
(c) Varian
ts identified for regioselective intramolecular C
-
H amination.
7
(d)
Variant identified for aziridination of styrenes.
8
Supplementary Table
2
.
Aminohydroxylation of 4
-
vinylanisole with
hemin and
various hemo
proteins
.
(a)
Catalyst
Note
Yield [%]
Whole cells expressing
pET22b(+)
pEC86 vector
pEC86 encodes ccmABCDEFG to aid the
maturation of cytochrome
c
proteins
0.8
Hemin
(b)
Concentration: 10 μM
0.1
Hemin + imidazole
(b)
10 μM hemin + 1 mM imidazole
0.08
Hemin +
bovine serum albumin
(b)
Concentration: 10 μM each
0.1
Bovine serum albumin only
Concentration: 10 μM
N. D.
Myoglobin
From equine heart, concentration: 10 μM
2.2
Rhodothermus marinus
(
Rma
)
cytochrome
c
4.0
(c)
Hydrogenobacter thermophilus
cytochrome
c
1.1
Rhodopila globiformis
cytochrome
c
1.0
Rma
cytochrome
c
M100D
2.9
Rma
cytochrome
c
“TDE”
(d)
Mutations from the wildtype: V75T M100D M103E
3.8
(a) Reactions
were
performed with 5 mM
of each substrate; results are the average of triplicate reactions. N.D. =
none detected.
W
hole cells
resuspended in M9
-
N medium to
OD
600
= 30
were generally used, unless otherwise
specified.
S
10
(b) Hemin and other additives were dissolved in M9
-
N medium wit
hout any whole cells to specified final
concentrations. Hemin was reduced using sodium dithionite solution (10 mg/mL) prior to reaction.
(c) Enantiomeric excess was measured for the product of wild
-
type
Rma
cytochrome
c
reaction. It was determined
to be 32
%
ee
.
(d) Variant identified for carb
e
ne Si
-
H insertion.
9
Supplementary Table
3
.
Aminohydroxylation data presented in Figure 2A of the main text
.
Variant
Mutations from the wild
-
type
Rma
cytochrome c
TTN
Yield [%]
ee [%]
Rma
cytochrome
c
None
40
4.8
32
“M100S”
M100S
56
5.3
39
“SG”
M100S, M103G
74
7.2
60
“SPG”
M100S, T101P, M103G
86
7.3
51
“VSPG”
M99V, M100S, T101P, M103G
360
15.0
73
VSPG T98L
T98L, M99V, M100S, T101P, M103G
640
25.8
76
VSPG M76Q T98L
M76Q,
T98L, M99V, M100S, T101P, M103G
1800
76.1
85
“TQL”
Y44T, M76Q, T98L, M99V, M100S, T101P, M103G
2500
90.4
90
Reactions
were
performed
using
whole cells at OD
600
= 30 with
10
mM of each substrate; results are the average
of
triplicate
reactions. Site
-
saturation
libraries were
generated and tested in the 96
-
well expression and reaction
format discussed in the general procedures. Beneficial mutations were identified, and the
best variants were
grown in 30 mL expression cultures and re
-
tes
ted.
The TTN and ee reported in this table are obtained with the
cultures grown
at
30
-
mL scale.
S
11
Supplementary Table
4
.
Summary of directed evolution for aminohydroxylation.
Generation
Parents
Saturated sites
Mutation
identified
1
Wildtype
Rma
cyt
c
M100X
M100S, M100H
2
M100S, M100H
M103X
M100S M103G (“SG”)
3a
“SG”
V75X, L105X
None
3b
“SG”
T101X
T101A, T101P
4a
“SG” T101A
M99X
T101P M99V (“VSPG”)
4b
“SG” T101P
5
“VSPG”
T98X
T98L
6
“VSPG” T98L
M76X, Y44X
M76Q
7
“VSPG” M76Q T98L
Y44X
Y44T
Reactions were performed in the 96
-
well expression and reaction format, with 10 mM of each screening
substrates.
Active site residues T98, M99, M100, T101, M103, and L105 are located in the loop distal to the heme
cofactor of
Rma
cytochrome
c
. Active site residues V75 and M76 are located on the helix on the distal face of
heme. Y44 is
near the
region where the active site is exposed to the aqueous environment
.