102
S. Banerjee et al. / Bioorganic Chemistry 62 (2015) 94–105
was located at a position almost equivalent to the Phe87 residue in
cytochrome P450BM-3 (CYP102A1), which has the closest similar-
ity with CYP175A1. Both of these two residues (e.g., Phe87 in cyto-
chrome P450BM-3, and Leu80 in CYP175A1) extend into the heme
pocket, and are positioned above the heme plane. Mutation of the
Phe87 residue in cytochrome P450BM-3 showed alteration of the
reaction specificity as well as of the enzymatic activity suggesting
important role of this residue in the substrate association to the
enzyme [17,50–54]. Therefore, we mutated Leu80 residue in the
CYP175A1 to phenylalanine residue (see Fig. 1b) that could allow
the planar aromatic substrate to access the distal site more easily
by aromatic interaction with Phe80 in the L80F mutant, and hence
could enhance the enzymatic efficiency (kcat/Km) as well as enzy-
matic activity (kcat).
3.2. Mutation of recombinant WT CYP175A1
The pKK-223 plasmid, encoding the WT CYP175A1 enzyme, was
a kind gift from V. B. Urlacher (University of Stuttgart, Stuttgart,
Germany). Site-directed mutagenesis was conducted using the
Quik-ChangeTM site-directed mutagenesis kit (Stratagene). The for-
ward and reverse oligonucleotide primers for the L80F mutant
were GGGAGGGGCCTCtTCACCGACTGGGG and CCCCAGTCGGTGAa-
GAGGCCCCTCCC, respectively. The mutation of the enzyme was
confirmed by DNA sequencing (see Fig. S15 in the Supporting
Information).
3.3. Protein expression and purification
The electronic environment of L80F in the heme active site and
the thermostability were found to be similar to those of the WT
enzyme (see Figs. S1, S12 and S13 and Supplementary Note 1 in
the Supporting Information). The kinetic studies showed that the
enzymatic activity for oxygenation of the substrates was slightly
faster in case of the L80F mutant compared to that in case of the
WT enzyme (see Table 2, Fig. 5b, Fig. S14, and Supplementary Note
2 in the Supporting Information).
The wild type CYP175A1 and its mutant (L80F) were expressed
and purified by earlier reported method [7,9,10,56]. In brief,
CYP175A1 was grown by taking a single colony of the BL21-DE3
codon plus RP cells, harboring the plasmid encoding the gene for
WT or mutant protein and inoculating it into the 2 ꢂ YT media con-
taining ampicillin (100 lg/mL) and chloramphenicol (50 lg/mL).
For the mutant protein, the heme precursor d-aminolevulinic acid
(50 M) was added to the culture 30 min before the induction of
l
Thus, the observed enhancement of the rate of product forma-
tion (Table 2) by the L80F mutant compared to the WT CYP175A1
possibly results from the enhanced affinity of the planar aromatic
substrates (see Table 1) at the enzyme pocket as discussed above.
However, the L80F mutation may also have other effects such as
decreased affinity of the hydroxylated product at the active site,
which could also reconcile the observed faster rate of oxygenation.
Although this mutation did not drastically increase the catalytic
efficiency of CYP175A1, the results obtained from the present
study indeed provide a direction to evolve suitable mutant enzyme
by protein engineering for efficient oxygenation of the aromatic
substrates in future.
protein expression with IPTG. Expression of protein was induced
by adding IPTG (1 mM final concentration), and the culture was
grown at 30 °C for 48 h. After the cell lysis, the protein was precip-
itated by (NH4)2SO4 (35–50% saturation), re-dissolved in buffer and
purified by a hydrophobic column (Phenyl Sepharose), and frac-
tions having Rz (Reinheitszahl, A417/A280) P 1.4 were collected.
The protein was further purified on a hydroxyapatite column and
fractions having Rz P 1.5 were collected. The purified fractions
were concentrated using centriprep concentrators (10 kD cut-off
membrane, Millipore), dialyzed in 50 mM KPi (pH 7.5) buffer and
stored at ꢁ25 °C in 50% glycerol. The concentration of CYP175A1
was estimated spectrophotometrically from the absorbance at
417 nm (e
417 = 104 mMꢁ1 cmꢁ1) [7]. The purity of the proteins
was checked by SDS–PAGE, and UV–visible absorption (see
Fig. S11 in the Supporting Information).
3. Experimental
The plasmids encoding wild type putidaredoxin (PdX) and puti-
daredoxin reductase (PdR) were kindly supplied by Prof. S.G. Sligar
(University of Illinois, USA). The wild type putidaredoxin (PdX) and
putidaredoxin reductase (PdR) were expressed and purified using a
reported procedure [57] and the concentration of PdX was deter-
3.1. Materials
The various components of bacteriological culture media were
purchased from Himedia, India. Ampicillin sodium salt, chloram-
phenicol, DNaseI (from bovine pancreas), PMSF, IPTG, d-
aminolevulinic acid (d-ALA), sodium cholate, anhydrous sodium
sulfate, potassium bicarbonate, NADH, horseradish peroxidase
(HRP), several naphthalene derivatives (substrates; see Table 1),
and oligonucleotide primers were purchased from Sigma–Aldrich.
Lysozyme (from hen egg white) was purchased from Fluka. The
Quik-ChangeTM site-directed mutagenesis kit was purchased from
Stratagene. Ammonium sulfate, dipotassium hydrogen ortho
phosphate, and potassium dihydrogen orthophosphate, tris–HCl
(2-amino-2-hydroxymethyl-propane-1, 3-diol hydrochloric acid),
and tris-base were purchased from USB Chemicals. The column
material Phenyl Sepharose was purchased from GE Healthcare
Bio-Sciences AB, Uppsala. Hydroxyapatite material was purchased
from Biorad Laboratories Inc, Hercules, CA. HPLC, and/or GC grade
methanol, chloroform, 2-propanol, n-hexane, and other required
solvents were purchased from Merck Chemicals. EDTA was pur-
chased from S.D. Fine Chemicals, India. All other buffer compo-
nents were purchased from Qualigens Fine Chemicals/S.D Fine
Chemicals, India. Millipore water was used for buffer preparation,
and for all other experimental work wherever required. Hydrogen
peroxide was purchased from Qualigens Fine Chemicals. The con-
centration of H2O2 was measured spectrophotometrically using
mined from the absorbance at 455 nm (
[58] and that of PdR was estimated from the absorbance at
454 nm (
454 = 10.9 mMꢁ1 cmꢁ1) [59].
e )
455 = 5.9 mMꢁ1 cmꢁ1
e
3.4. Enzymatic oxygenation of the substituted naphthalenes
Oxygenation/oxidation of substituted naphthalene (see Table 1)
by CYP175A1 was carried out by incubating 500
lL reaction mix-
ture containing 10 CYP175A1, 500 substituted naph-
lM
lM
thalene (substrate) and 10 mM H2O2 in 50 mM KPi buffer
(pH = 7.5) at 50 °C unless otherwise mentioned. The controls for
the reactions were carried out that contained all the reactants
except CYP175A1 or except H2O2 in the assay mixture. Each reac-
tion was allowed to proceed for 60 min unless otherwise stated.
The reaction mixture was then extracted with 500
(containing 10% methanol) and the chloroform layer was collected,
dried over anhydrous sodium sulfate, filtered through 0.2 m syr-
lL chloroform
l
inge filter. The extracted reaction mixtures were then analyzed by
different mass spectrometry techniques.
The kinetic parameters for the enzymatic reaction were deter-
mined by initial rate method at 50 °C by mixing 10 mM of H2O2
the molar extinction coefficient (
[55].
e
) 39.4 Mꢁ1 cmꢁ1 at 240 nm
to a 500
lL solution containing different concentrations of the sub-
strate (varied between 100 and 500
l
M) and 10 M CYP175A1 in
l