CHEMBIOCHEM
FULL PAPERS
hemoproteins, including cytochrome P450s, and can be im-
proved by protein engineering.
Hemochrome assay for whole cells: For whole-cell reactions, he-
mochrome assays were performed as follows: cells were normal-
ized to OD =30 in lysis buffer (25 mm Tris·HCl, 10 mm NaCl,
We hypothesized that different active site shapes would in-
fluence the stereochemical mixture of products formed. Con-
sistent with this hypothesis, we found that both the choice of
parent enzyme and the axial ligand influenced the activity and
stereoselectivity of cyclopropanation. Thus, we propose that
variants of different cytochrome P450s, and possibly other he-
moproteins, will be interesting catalysts for the chemical syn-
thesis of stereochemically complex cyclopropanated products.
Serum albumins show a high affinity for metal porphyrins
and provide a hydrophobic pocket that allows them to cata-
lyze this non-natural reaction as well as any of the wild-type
P450s. The difference, we believe, lies in the potential for evo-
lution. Albumins are expressed as insoluble aggregates in
600
À1
À1
0
.5 mgmL lysozyme and 0.02 mgmL DNaseI). Cell lysis was per-
formed for 2 h at 378C with an agitation rate of 200 rpm. The
resulting lysate was centrifuged for 10 min at 20000g, and the su-
pernatant was subjected to the hemochrome assay. The final
sample volume was adjusted with H O (to 735 mL). A solution of
2
NaOH (1m, 75 mL) was added, and the solution was incubated for
5 min at room temperature. Pyridine (175 mL) was added, and the
solution was mixed via pipetting. A solution of sodium dithionite
À1
(
100 mgmL , 15 mL) was added and the solution was sealed and
incubated for 10 min. Samples were measured by using UV/Vis.
Heme concentrations were determined by using the reported ex-
À1
À1 [20]
tinction coefficient (e=196000m cm ).
Protein expression: All enzymes were expressed in BL21(DE3)
E. cloni (Lucigen). Depending on the plasmid, cultivation was per-
[
16]
E. coli,
preventing simple evaluation of these proteins as
À1
formed with kanamycin sulfate (50 mgmL ) for pET28 or ampicillin
whole-cell catalysts and making it impossible to engineer or
direct their evolution in E. coli. Cytochrome P450s, in contrast,
have a defined substrate-binding pocket and are highly evolva-
ble, features that lead to their ready adaptation to new catalyt-
ic demands.
À1
sodium salt (100 mgmL ) for pET22. LB medium (50 mL) was ino-
culated with a single clone, and the overnight culture was used to
inoculate Hyper Broth (final volume: 750 mL) with d-ALA (250 mm
final concentration) and incubated at 378C with a 225 rpm agita-
tion rate. At OD600 =1.5, cultures were induced with IPTG (500 mm
final concentration), the temperature was lowered to 208C, the agi-
tation rate was lowered to 120 rpm, and the culture was incubated
for 24 h. Following expression, cells were pelleted and frozen at
À208C. For whole-cell catalysis, cells were resuspended in in nitro-
gen-free M9 medium (1 L contains 31 g Na HPO , 15 g KH PO ,
Experimental Section
Unless otherwise noted, all chemicals and reagents were obtained
from commercial suppliers (Sigma–Aldrich, Acros). Gas chromatog-
raphy analyses were carried out on a Shimadzu GC-17A gas chro-
matograph, an FID detector, and J&W Scientific (Folsom, CA) cyclo-
sil-B columns (30 mꢁ0.32 mm, 0.25 mm film and 30 mꢁ0.25 mm,
2
4
2
4
2
.5 g NaCl, 0.24 g MgSO , and 0.01 g CaCl ).
4 2
Protein purification: For purification, frozen cell pellets were re-
À1
suspended (4 mLg
wet cell weight) in lysis buffer (25 mm
0
.25 mm film). pET22 and pET28 plasmids were used as cloning and
À1
Tris·HCl, 100 mm NaCl, 10 mm imidazole, lysozyme (0.5 mgmL ),
expression vectors. All gene constructs except P450BM3 and
CYP153A6 were generated from codon-optimized gBlocks (Inte-
grated DNA Technologies (IDT), San Diego, CA). Site-directed muta-
genesis was accomplished by standard overlap mutagenesis with
primers bearing desired mutations (IDT). Gene and primer sequen-
ces are available upon request. Electrocompetent BL21(DE3)
E. cloni (Lucigen, Middleton, WI) cells were prepared following the
À1
À1
DNaseI (0.02 mgmL ), hemin (1 mgg wet cell weight), pH 7.0).
À1
TxtE was resuspended (4 mLg wet cell weight) in lysis buffer I
(25 mm Tris·HCl, 100 mm NaCl, and 30 mm imidazole, pH 7.0). Cells
were then disrupted by sonication (3 min, output control: 1.5, duty
cycle: 5 s on/10 s off; Sonicator 3000, Misonix, Inc.). In the case of
the thermostable CYP119, the cell suspension was subsequently
incubated for 30 min at 608C to precipitate E. coli proteins and im-
prove purification. To pellet insoluble cell debris, lysates were cen-
trifuged (30000g for 30 min at 48C). Cleared lysates were filtered
through sterile cellulose acetate syringe filters (0.22 mm, VWR) and
then purified with Ni-NTA columns (GE Healthcare or Roche Ap-
plied Science) by using an AKTAxpress purifier FPLC system (GE
Healthcare). All enzymes were eluted with a linear gradient from
[17]
protocol of Sambrook et al. Restriction enzymes, Phusion poly-
merase, and T4 ligase were purchased from New England Biolabs
(
(
NEB, Ipswich, MA). Alkaline phosphatase was obtained from Roche
Nutley, NJ). Gibson cloning reactions were performed according to
the manufacturer’s recommendations. Hyper Broth was obtained
from AthenaES and used according to the manufacturer’s recom-
mendations.
1
00% buffer A (25 mm Tris·HCl, 100 mm NaCl, 10 mm, pH 7.0) or
buffer A1 for TxtE (25 mm Tris·HCl, 100 mm NaCl, 30 mm imidazole,
pH 7.0), 0% buffer B (25 mm Tris·HCl, 100 mm NaCl, 400 mm imida-
zole pH 7.0) or buffer B1 for TxtE (25 mm Tris·HCl, 100 mm NaCl,
Determination of enzyme concentration: Enzyme concentrations
were determined from ferrous carbon monoxide binding difference
spectra by using previously reported extinction coefficients for cys-
À1
À1 [18]
3
00 mm imidazole, pH 7.0) to 100% buffer B over 10 column vol-
teine-ligated (e=91000m cm )
and serine-ligated enzymes
e=103000m cm ). Concentrations of the serine-ligated mu-
tants were also determined via hemochrome assay by using the
À1
À1 [19]
umes. Fractions containing the respective enzymes were pooled,
concentrated, and exchanged into storage buffer (25 mm Tris·HCl,
(
À1
À1 [20]
1
0 mm NaCl, pH 7.5) or (20 mm Tris·HCl, pH 7.5) for TxtE. Concen-
reported extinction coefficient (e=196000m cm ).
trated proteins were aliquoted, flash-frozen on powdered dry ice,
and stored at À208C. Enzyme concentrations were determined via
CO binding difference spectra as described above from frozen ali-
quots.
Hemochrome assay: The final sample volume of purified protein
samples was adjusted with H O (to 735 mL). A solution of NaOH
2
(
1m, 75 mL) was added and incubated for 5 min at room tempera-
ture. Pyridine (175 mL) was added, and the solution was mixed via
À1
pipetting. A solution of sodium dithionite (100 mgmL , 15 mL)
In vitro small-scale bioconversions under anaerobic conditions:
Small-scale reactions (400 mL, final enzyme concentration: 10 mm)
were conducted in crimp vials (2 mL size, Agilent Technologies,
San Diego, CA). A solution of the P450 enzyme (26.6 mm enzyme in
storage buffer, 135 mL) and KPi buffer (1m, pH 8.0, 15 mL) was
was added and the solution was sealed and incubated for 10 min.
Samples were measured by using UV/Vis spectroscopy. The heme
concentration was determined by using the reported extinction co-
À1
À1 [20]
efficient (e=196000m cm ).
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 8
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These are not the final page numbers!
Heel, Thomas
