Modification of an Enzyme Biocatalyst for the Efficient and Selective Oxidative Demethylation of para-Substituted Benzene Derivatives

Article abstract of DOI:10.1002/cctc.201600951

The bacterial CYP199A4 enzyme is able to oxidise a narrow range of aromatic acids, which includes 4-methoxybenzoic acid, efficiently. A serine 244 to aspartate variant was identified with enhanced activity for a wide range of para-methoxy-substituted benzenes. Substrates in which the acidic benzoic acid moiety is replaced with a phenol and the amide, aldehyde and bromide analogues were all oxidised with high activity by the S244D mutant (product formation rate >600 nmol nmol_CYP ^?1 min^?1) with turnover numbers of up to 20 000. If the carboxylate moiety was modified to a nitro, ketone, boronic acid, hydroxymethyl or nitrile group, these substrates were also oxidised at a significantly higher activity by S244D than the wild-type enzyme. 3,4-Dimethoxybenzaldehyde was demethylated selectively and oxidatively to 3-methoxy-4-hydroxybenzaldehyde by the S244D mutant 84-fold more rapidly than with the wild-type enzyme. CYP199A4 would have applications in the catalytic regioselective oxidative demethylation of suitably substituted benzene substrates under mild conditions and in the presence of more oxidatively sensitive functional groups, such as an aldehyde.

Full text of DOI:10.1002/cctc.201600951

DOI: 10.1002/cctc.201600951
Full Papers
Modification of an Enzyme Biocatalyst for the Efficient and
Selective Oxidative Demethylation of para-Substituted
Benzene Derivatives
Rebecca R. Chao,[a] Ian C.-K. Lau,[a] James J. De Voss,*[b] and Stephen G. Bell*[a]
The bacterial CYP199A4 enzyme is able to oxidise a narrow
range of aromatic acids, which includes 4-methoxybenzoic
acid, efficiently. A serine 244 to aspartate variant was identified
with enhanced activity for a wide range of para-methoxy-sub-
stituted benzenes. Substrates in which the acidic benzoic acid
moiety is replaced with a phenol and the amide, aldehyde and
bromide analogues were all oxidised with high activity by the
S244D mutant (product formation rate >600 nmoln-
molCYPꢀ1 minꢀ1) with turnover numbers of up to 20000. If the
carboxylate moiety was modified to a nitro, ketone, boronic
acid, hydroxymethyl or nitrile group, these substrates were
also oxidised at a significantly higher activity by S244D than
the wild-type enzyme. 3,4-Dimethoxybenzaldehyde was deme-
thylated selectively and oxidatively to 3-methoxy-4-hydroxy-
benzaldehyde by the S244D mutant 84-fold more rapidly than
with the wild-type enzyme. CYP199A4 would have applications
in the catalytic regioselective oxidative demethylation of suita-
bly substituted benzene substrates under mild conditions and
in the presence of more oxidatively sensitive functional
groups, such as an aldehyde.
Introduction
The cytochromes P450 are a family of versatile enzymes that
catalyse the insertion of an O atom from O2 into the CꢀH
bonds of organic molecules primarily.[1] In addition to this ac-
tivity, these enzymes have found widespread use in nature as
catalysts for other reactions that include heteroatom oxidation
desaturation, rearrangements, CꢀC bond cleavage and cou-
pling reactions.[2] CYP199A4 from the Rhodopseudomonas pal-
ustris strain HaA2 is a heme-dependent cytochrome P450 (CYP)
monooxygenase enzyme that is able to demethylate 4-me-
thoxybenzoic acid efficiently.[3] Mechanistically, the O-demethy-
lation of 4-methoxybenzoic acid occurs through hydrogen ab-
straction by Compound I,[4] followed by oxygen rebound to
give the hydroxylated hemiacetal.[5] The hemiacetal decompos-
es spontaneously to yield 4-hydroxybenzoic acid and formalde-
hyde.[2b,6] The high enzymatic activity of CYP199A4 is support-
ed by a class I electron transfer chain from the same bacteri-
um, which consists of a [2Fe-2S] ferredoxin (HaPux) and
a flavin-dependent ferredoxin reductase (HaPuR) and mediates
heme reduction by NADH.[3,7] A whole-cell system, which con-
tains CYP199A4 and its electron transfer partners, has been
constructed that is capable of product formation on a gram-
per-litre scale.[3,6b]
The stable, soluble nature and high activity of bacterial CYP
enzymes, such as CYP199A4, makes them of interest as cata-
lysts in synthetic chemistry that involves CꢀH bond oxidation.[8]
However CYP199A4 is only able to oxidise a narrow range of
substrates, and 4-methoxybenzoic acid induces an optimal ac-
tivity. The alteration of the carboxylate group or modification
of the benzene ring both have an adverse effect on substrate
binding and activity.[9] However, the replacement of the me-
thoxy moiety with alternative groups or the addition of small
substituents at the 2- or 3-positions is somewhat tolerated.[6b,9]
For instance, CYP199A4 can hydroxylate 4-methylbenzoic acid
at the methyl group and O-demethylate 2,4- and 3,4-dimethox-
ybenzoic acids with total regioselectivity to generate 2-me-
thoxy- and 3-methoxy-4-hydroxybenzoic acids, respectively.[3,6b]
Previously, we have shown that the substrate carboxylate
moiety is critical for substrate binding to CYP199A4. The altera-
tion of the benzoic acid to a benzyl alcohol, benzamide, ben-
zaldehyde or even phenylacetic acid all had a significant
debilitative effect on binding.[9b,10,11]
Several crystal structures of the substrate-free and substrate-
bound forms of CYP199A4 and its close homologue CYP199A2,
from the CGA009 strain of R. palustris, have been deter-
mined.[6a,9b,12] In the crystal structures of the 4-methoxybenzoic
acid bound enzymes (PDB codes 4DO1 and 4DNJ), the para
substituent is held over the heme iron centre. This is consistent
with the exclusive attack at the group para to the carboxylate.
The substrate carboxylate interacts directly with the side
chains of arginine 92, serine 95 and serine 244 and, through
a bridging water molecule, arginine 243 (Figure 1).[12] In addi-
tion, a chloride anion is co-opted by the enzyme and closes off
[a] R. R. Chao, I. C.-K. Lau, Dr. S. G. Bell
Department of Chemistry
University Adelaide
Adelaide, SA, 5005 (Australia)
E-mail: stephen.bell@adelaide.edu.au
[b] Prof. J. J. De Voss
School of Chemistry and Molecular Bioscience
University of Queensland
St Lucia, Qld, 4072 (Australia)
E-mail: jdevoss@uq.edu.au
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cctc.201600951.
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in the substrate binding pocket of the CYP199A4 enzyme. The
residues chosen for investigation included: arginine 243,
serine 95 and serine 244, which interact directly, or through
a bridging water molecule, with the carboxylate group; pro-
line 94 located at the entrance of the substrate-access channel;
and phenylalanine 182, which defines the substrate binding
pocket. The S244D mutant was shown to have greatly en-
hanced activity for a wide range of para-substituted benzenes,
whereas the S244N and other variants do not.
Results
Screening of mutant forms of CYP199A4
In total, 10 mutants of CYP199A4 were constructed and tested
for oxidation activity against a selection of substrates in which
the carboxylate group of 4-methoxybenzoic acid had been
modified: P94V, S95A, S95D, S95N, F182L, R243L, R243T, S244A,
S244D and S244N (Figure 1). These residues were chosen be-
cause of their location in the active site. Serine 95, arginine 243
and serine 244 interact with the carboxylate group of the sub-
strate. To assess the effect of different mutations, the charged
arginine 243 was altered to a neutral leucine and polar threo-
nine, and the two serine residues were changed to hydropho-
bic alanine, acidic aspartate and amide asparagine residues.
Proline 94, in the substrate-access channel, was altered to
valine, and phenylalanine 182, which contacts the benzene
ring of the substrate, was engineered to a leucine, as controls.
Mutations of the equivalent phenylalanine residue in
CYP199A2 have been reported to improve cinnamic acid
oxidation.[17]
Figure 1. Active site of 4-methoxybenzoic acid bound CYP199A4 (PDB code
4DO1). The amino acid residues that confer the substrate specificity of the
enzyme are shown. 4-Methoxybenzoic acid is shown in yellow, the heme in
blue, serine 244 in magenta, the other amino acids in green and the
bridging water molecule in red.[12]
the access channel. This chloride anion interacts with argi-
nine 92 as well as tyrosine 177 and glutamine 203. These
enzyme–substrate contacts account partially for the substrate
specificity of the enzyme, and variants with modifications of
the residues at these positions show a dramatically weakened
affinity for 4-methoxybenzoic acid and a reduction in the
enzyme activity.[12]
The mutant genes of CYP199A4 were cloned into the
pRSFDuetHaPux vector and used in conjunction with pETDue-
tHaPuR/HaPux for the whole-cell screening of substrate oxida-
tion using small-scale, low-cell-density E. coli cultures (Experi-
mental Section).[3] Four substrates, 4-methoxybenzoic acid (1),
4-methoxyphenol (2), 4-methoxyacetophenone (3) and 4-me-
thoxynitrobenzene (4) were chosen for the initial screening
process (Figure 2). These substrates were selected based on
their different functionalities para to the methoxy group, and
their likely products were available for facile identification by
coelution experiments. In addition, the yellow 4-nitrophenol
product that arises from the O-demethylation of 4-methoxyni-
trobenzene gives a visual indication of the progress of sub-
strate oxidation. The results should provide an indication of
the likely activity of the mutants towards carboxy-modified
substrates.
The related CYP199A2 enzyme has been reported to be
a biocatalyst for the oxy-functionalisation of various other aro-
matic carboxylic acids.[6a,13] These enzymes can be engineered
to enhance their activities and broaden their substrate
range.[14] Recently, mutant forms of CYP199A2 have been
shown to be catalysts for the oxidation of benzyl alcohols and
phenols. However, a detailed analysis of the substrate binding
and enzyme kinetics was not presented as the experiments
were performed by using a whole-cell oxidation system.[15]
Knowledge of the critical enzyme–substrate interactions is of
paramount importance to facilitate efforts to engineer enzyme
systems rationally to achieve the turnover numbers required
for large-scale synthetic applications.[7a,8b,14a,b,d,15,16] Previously,
we have shown that the R243T, R92E and S95V mutants all
have a significant debilitative effect on 4-methoxybenzoic acid
binding to CYP199A4 and reduce the activity of the enzyme.[12]
Here, we report the expansion of the substrate range of
CYP199A4 through the mutation of a single amino acid,
serine 244, in the active site of the enzyme. This variant was
identified by screening 10 mutants designed with substitutions
The oxidation activities of the majority of the mutants for 4-
methoxybenzoic acid remained high. After 3 h, no chromato-
graphically detectable substrate remained in the whole-cell
turnovers of 4-methoxybenzoic acid that contained the P94V,
S95A, R243L, R243T, S244A and S244D variants, and most of
the substrate had been oxidised to product in 3 h in the S95N
turnover. Small amounts of 4-hydroxybenzoic acid were ob-
served in the S244N whole-cell oxidations after 3 h, and more
product was observed after an additional 15 h (Figure S1). Only
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Figure 2. Substrates tested for in vitro activity with WT CYP199A4 and the
S244D and S244N variants. The four substrates on the top row were used to
screen all the variants using whole-cell oxidation systems.
trace quantities of 4-hydroxybenzoic acid were generated by
the S95D and F182L variants even after 18 h.
The oxidation activity observed with the majority of the mu-
tants for 4-methoxyacetophenone was considerably lower.
Only in the S244D turnover was a large amount of product
generated after 3 h, and ꢁ90% conversion was observed (Fig-
ure 3a). A small amount of 4-hydroxyacetophenone was pro-
duced by S244A during this time (<15%) and even smaller
quantities were observed in the whole-cell oxidations by S95A,
R243L and R243T. Very low levels of 4-hydroxyacetophenone
were detected in the turnovers using P94V, S95D, S95N, F182L
and S244N. After 18 h, only small increases in the amount of
product formed were observed for these CYP199A4 variants,
but almost all the substrate was converted to product in the
S244D mutant (Figure S2a).
Figure 3. a) HPLC analysis of the whole-cell oxidation of 4-methoxyaceto-
phenone (tR =15.0 min) to 4-hydroxyacetophenone (tR =8.0 min) by
CYP199A4 variants after 3 h. For clarity, the chromatograms are offset along
the x and y axes. b) UV/Vis spectra of the supernatant from 4-methoxynitro-
benzene oxidation to the yellow 4-nitrophenol product by the CYP199A4
variants after 3 h.
comparison of the total combined product levels of each sub-
strate revealed that the activity of the other mutants for the
carboxy-modified substrates was much lower in the order:
S244A>S95N>R243LꢁR243T>P94VꢁS244NꢁS95Aꢁ
S95D>F182L. Although the different CYP199A4 variants may
be produced at different levels in the whole-cell system, the
S244D variant generated significantly higher levels of product
than all the other mutants tested (Figure 3 and Figures S2 and
S3). The WT CYP199A4 enzyme has also been shown to have
a low activity with these substrates in vitro and in vivo.[10]
Therefore, S244D was selected for further study with a range
of carboxy-modified substrates (Figure 2). For comparison and
to validate the reliability of our whole-cell screening process,
the S244N mutant, which demonstrated only modest oxidation
activity, was also investigated.
The S244D variant also exhibited a noticeably higher oxida-
tion activity with 4-methoxyphenol compared to the other mu-
tants and converted all of the substrate to product in the first
3 h (Figure S2b). Considerably lower (ꢁ10% conversion) levels
of product were formed by F182L and S95D in this time. Mini-
mal levels of product, if any, were detected in the remaining
turnovers after 3 h, and a low conversion of substrate to prod-
uct occurred in the whole-cell oxidations after a further 15 h.
In the colorimetric assay of 4-methoxynitrobenzene turn-
overs, noticeably more yellow 4-nitrophenol product was ob-
served in the supernatant of the system that incorporated the
S244D mutant. UV/Vis spectrophotometry indicated that more
than three times as much 4-nitrophenol was formed in the first
3 h by S244D compared to the other turnovers (Figure 3b).
The other whole-cell reactions were visibly paler in colour com-
pared to that of S244D. After 18 h, the S244A turnover gener-
ated comparable levels of product to that of S244D, but the
product concentrations of the remaining whole-cell oxidations
were less than half that of S244D (Figure S3).
Activity and product formation assays with the S244D and
S244N mutants
In all the in vitro turnovers, the products observed arose exclu-
sively from demethylation of the para-methoxy group
(Scheme 1, Figures S4–S6). In the turnover of 4-methoxybenzo-
ic acid, S244D oxidised NADH at less than half the rate of the
wild-type (WT) enzyme (566 minꢀ1). However, the coupling of
reducing equivalents to product formation in the reaction re-
mained high (coupling efficiency 82%), which resulted in the
These results indicate that the S244D mutant of CYP199A4
exhibited the highest oxidation activity with 4-methoxyaceto-
phenone, 4-methoxyphenol and 4-methoxynitrobenzene. A
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Table 1. Catalytic turnover activity data for WT CYP199A4 and the S244D
and S244N variants with various substrates. The data are given as the
mean ꢂ standard deviation (SD) with nꢃ3. The reaction mixtures (50 mm
Tris, pH 7.4) contained 0.5 mm P450, 5 mm HaPux and 0.5 mm HaPuR. Rates
are given as nmolnmolCYPꢀ1 minꢀ1. The data for the WT enzyme with sev-
eral substrates were reported previously and are included for compari-
son.[3,9b,10]
Substrate
N[a]
PFR[b]
C [%][c]
TON[d]
TTN[e]
1-WT[3]
1340ꢂ28
566ꢂ2
26ꢂ2
8ꢂ0.3
841ꢂ56
20ꢂ1
9.1ꢂ0.2
160ꢂ7
11ꢂ1
–
1219ꢂ120
460ꢂ27
15ꢂ1
91ꢂ2
82ꢂ4
58ꢂ1
–
535
400
–
544
–
–
1-S244D
1-S244N
2-WT[10]
2-S244D
2-S244N
3-WT[10]
3-S244D
3-S244N
4-WT[10]
4-S244D
4-S244N
5-WT[10]
5-S244D
5-S244N
6-WT[9b]
6-S244D
6-S244N
7-WT[9b]
7-S244D
7-S244N
8-WT[10]
8-S244D
8-S244N
20000
–
–
0.7ꢂ0.1
600ꢂ35
2.4ꢂ0.4
1.4ꢂ0.1
140ꢂ3
9ꢂ0.5
72ꢂ4
12ꢂ1
15ꢂ1
85ꢂ2
23ꢂ2
71ꢂ3[f]
81ꢂ4[f]
21ꢂ1[f]
13ꢂ2
100ꢂ5
38ꢂ2
17ꢂ1
99ꢂ2
34ꢂ2
21ꢂ2
54ꢂ1
24ꢂ3
27ꢂ2
42ꢂ2
26ꢂ2
14700
–
–
–
552
172
–
–
–
8100
Scheme 1. Products formed from CYP199A4 enzyme turnovers.
2.5ꢂ0.4
14ꢂ2[f]
–
–
–
–
–
–
270ꢂ14[f]
2.5ꢂ0.1[f]
1.8ꢂ0.1
1050ꢂ52
4.8ꢂ0.3
3.0ꢂ0.1
860ꢂ33
2.1ꢂ0.1
4.0ꢂ0.6
109ꢂ2
formation of 4-hydroxybenzoic acid at 463 minꢀ1. The NADH
oxidation and product formation rates of S244N with 4-me-
thoxybenzoic acid were 50- and 80-fold slower than that of WT
CYP199A4 (26 and 15 minꢀ1, respectively; Table 1). The cou-
pling efficiency of the reaction, which is the proportion of
product formed versus NADH consumed, was also reduced
(58%; Figure S4a).
15ꢂ2
1050ꢂ6
12ꢂ0.2
15ꢂ2
868ꢂ32
6.3ꢂ0.5
19ꢂ1
202ꢂ2
10ꢂ1.0
15ꢂ1
131ꢂ8
11ꢂ1
–
–
640
276
–
648
250
–
358
182
–
281
162
20000
–
–
20000
–
–
9100
The activity of S244D was highest with 4-methoxybenza-
mide, for which the NADH oxidation and product formation
rates were improved relative to that of the WT enzyme by 70-
and 600-fold, respectively (Table 1). The coupling of redox
equivalents to product formation was almost complete and re-
sulted in the production of 4-hydroxybenzamide at a rate of
1050 minꢀ1 (Figure 4). The rate of NADH oxidation by S244N
with 4-methoxybenzamide was comparable to that of the WT
enzyme, but product formation was slightly faster (4.8 vs.
3.0 minꢀ1) because of the higher coupling efficiency of the
reaction (38 vs. 13%; Table 1).
2.4ꢂ0.3
4.0ꢂ0.5
55ꢂ6
–
–
–
–
2.8ꢂ0.4
[a] N: NADH turnover rate. [b] PFR: Product formation rate. [c] C: Coupling
efficiency, which is the percentage of NADH consumed in the reaction
that led to the formation of products. [d] TON: Average turnover number
for the NADH consumption assay (the maximum TON possible was 640–
700). [e] TTN: Maximum total turnover number observed using assays set
up like those above with 0.1 mm CYP enzyme and 2 mm substrate (theo-
retical maximum 20000). [f] Determined by monitoring the p-nitropheno-
late formed using A410
.
CYP199A4 enzyme (Table 1, Figures S4 and S6). This resulted in
product formation rates that ranged from 140 minꢀ1 for 4-me-
thoxyacetophenone to 860 minꢀ1 for 4-methoxybenzaldehyde.
The catalytic turnovers of 4-methoxybenzyl alcohol (7) and 4-
methoxyphenylboronic acid (8) were also improved by S244D,
albeit to a lesser extent, with a 20- and 14-fold increase in the
product formation activities, which were 109 and 55 minꢀ1, re-
spectively (Table 1). As with 4-methoxybenzamide, the im-
proved catalytic activity and levels of product formation were
because of increased rates of NADH oxidation and higher
levels of coupling efficiency (Table 1). No improvement or only
a small increase in activity was observed with the S244N
variant in all cases (Table 1).
Figure 4. Respective NADH oxidation and product formation rates of WT
CYP199A4 (blue and cyan) and S244D (green and lime) with selected car-
boxy-modified substrates. The activity of S244N is comparable with that of
WT CYP199A4 (Table 1).
The rates of NADH oxidation with 4-methoxynitrobenzene
could not be determined as the substrate absorbs strongly at
l=340 nm. However, as the oxidation product 4-nitrophenol
absorbs at l=410 nm, this wavelength was used to measure
the product formation rate by using UV/Vis spectrophotometry
(Figure S7). The product formation rate of S244N was slower
than that with the WT enzyme (2.5 vs. 14 minꢀ1) as was the
Similar large improvements in the product formation activity
of the S244D variant with 4-methoxybenzaldehyde (6; 300-
fold), 4-methoxyacetophenone (100-fold) and 4-methoxyphe-
nol (900-fold) were observed compared to that of the WT
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coupling efficiency of the reaction (21 vs. 71%). Although only
a modest increase in coupling was observed with S244D
(81%), the rate of product formation was approximately
20 times faster (270 minꢀ1; Table 1, Figure 4, Figure S7).
Table 2. Substrate binding parameters and catalytic turnover activity
data for WT CYP199A4 and the S244D variant with various substrates.
The data are given as the mean ꢂ SD with n ꢃ3. The turnovers were
performed as outlined in Table 1.
Overall, the results show a significant improvement in the
oxidation activity of S244D relative to WT CYP199A4 (Figure 4)
with a variety of para-substituted methoxybenzenes, whereas
little to no improvement was observed for S244N (Table 1).
Both the S244D and S244N variants of CYP199A4 gave almost
complete shifts to l=450 nm after reduction and the addition
of CO, which implies that both were fully functional P450 en-
zymes (Figure S8). Importantly, the different oxidation rates ob-
served in vitro reflect the relative activity of S244D and S244N
from the initial whole-cell screening studies, and S244D was
superior to the S244N variant.
Substrate
N[a]
PFR[b]
C [%][c]
9-WT
9-S244D
10-WT
10-S244D
11-WT
11-S244D
12-WT
12-S244D
13-WT
13-S244D
14-WT
14-S244D
15-WT
15-S244D
20ꢂ1
3.8ꢂ0.2
33ꢂ2
19ꢂ1[d]
37ꢂ2[e]
80ꢂ8[f]
90ꢂ8[g]
2ꢂ0.3
16ꢂ1
89ꢂ1
102ꢂ2
773ꢂ27
10ꢂ0.4
55ꢂ3
82ꢂ7
690ꢂ34
0.2ꢂ0.03
9.5ꢂ0.5
2.9ꢂ0.2
12ꢂ2
15ꢂ1
19ꢂ1
18ꢂ1
67ꢂ6
16ꢂ1
1.7ꢂ0.2
16ꢂ0.8
2.1ꢂ0.2
42ꢂ1
11ꢂ1
51ꢂ3
33ꢂ1
16ꢂ0.3
99ꢂ3
12ꢂ1
43ꢂ2
9.3ꢂ0.3
10ꢂ1
0.1ꢂ0.1
8.4ꢂ0.7
0.9ꢂ0.01
83ꢂ6[h]
Further activity studies of the S244D variant of CYP199A4
[a] N: NADH turnover rate. [b] PFR: Product formation rate. [c] C: Coupling
efficiency; the percentage of NADH consumed in the reaction that led to
the formation of products. [d] TON: 121/641. [e] TON: 262/696. TTN: 1100.
[f] TON: 558/670. [g] TON: 570/645. TTN: 13000. [h] TTN: 870.
As the S244D variant of CYP199A4 was active with the above
substrates, this enzyme was investigated with additional car-
boxy-modified derivatives (Figure 5). These were also tested
with WT CYP199A4 to determine the relative improvement in
oxidation activity obtained with the aspartate mutant.
of 4-cyanophenol (Table 2, Figures S4 and S5). The oxidative
demethylation of 4-methoxy-1-bromobenzene (10) by WT
CYP199A4 was tightly coupled (80%) and proceeded relatively
quickly at 82 minꢀ1. The same substrate was oxidised to 4-bro-
mophenol even more rapidly by S244D at a product formation
rate of 690 minꢀ1 (Table 2, Figures S5 and S6). An improvement
in the rate of 1,4-dimethoxybenzene (11) oxidation with S244D
was observed (9.5 vs. 0.2 minꢀ1; Table 2). However, hydroqui-
none, not 4-methoxyphenol, was the sole product detected in
the S244D reaction, which suggests the further oxidation of
the initial singly demethylated product (Figures S5 and S6).
This can be rationalised as 4-methoxyphenol is a more favoura-
ble substrate for S244D than WT CYP199A4 based on its rapid
oxidation to hydroquinone by this enzyme (600 minꢀ1; Table 1).
Increases in the activity of S244D relative to that of WT
CYP199A4 were observed with the methyl ester of 4-methoxy-
benzoic acid 12 and 4-methoxy-1-trifluoromethoxybenzene
(13). However, the overall product formation rates were rela-
tively low compared to that of the other substrates tested
(Tables 1 and 2).
Moderate increases in product formation activity were ob-
served in the turnover of 4-methoxybenzonitrile (9) with
S244D, which resulted in a 10-fold increase in the generation
The S244D variant oxidised 4-methoxytoluene (14) 20 times
faster than the WT enzyme (Table 2). The turnover of this sub-
strate was interesting in that it gave rise to two products iden-
tified as 4-methoxybenzyl alcohol and 4-hydroxybenzyl alcohol
(Figures S5 and S6). The doubly oxidised metabolite, 4-hydrox-
ybenzyl alcohol, was the major product, which formed 89 and
78% of the total product distribution for the WT and S244D
turnovers, respectively. The minor product, 4-methoxybenzyl
alcohol, must arise if the substrate is bound with the methoxy
group bound in the active site pocket normally occupied by
the carboxylate group with the methyl group held over the
heme iron centre.
To assess the requirement and selectivity of the S244D var-
iant for the para substituent (Scheme 1), we tested the disub-
Figure 5. Substrates tested for binding and activity with WT CYP199A4 and
the S244D variant.
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stituted substrate 3,4-dimethoxybenzaldehyde (15). The addi-
tion of this substrate to the WT CYP199A4 and the aspartate
mutant did not result in any appreciable increase in the rates
of NADH oxidation above the leak rates of the enzymes. In the
WT turnover, the lack of coupling of redox equivalents to prod-
uct formation (ꢁ1%) resulted in only trace amounts of 3-me-
thoxy-4-hydroxybenzaldehyde (Scheme 1, Figures S4 and S5).
The S244D reaction was, however, much more tightly coupled
(83%) to produce 3-methoxy-4-hydroxybenzaldehyde at a rate
of 8.4 minꢀ1. In addition, we tested phenol and 2- and 3-me-
thoxyphenol with the CYP199A4 variants. None of these sub-
strates generated any detectable product from the in vitro
turnovers (data not shown). These results and the efficient oxi-
dation of 3,4-dimethoxybenzaldehyde to only 3-methoxy-4-hy-
droxybenzaldehyde further demonstrate the selectivity of
CYP199A4 for demethylation solely at the para position.
The turnovers of the N-containing substrates 4-methoxyami-
nobenzene (16), N-(4-methoxyphenyl)acetamide (17) and 4-
methoxy-N,N-dimethylaniline (18) did not yield any detectable
product. The acetic acid ester derivative, 4-methoxybenzyl ace-
tate (19), was also tested with the S244D and WT CYP199A4
enzymes, but the substrate underwent ester hydrolysis under
the reaction conditions, and no product was observed during
the turnover of methyl 4-methoxyphenylacetate (20) with
either enzyme.
Table 3. Selected substrate binding data for WT CYP199A4 and the
S244D variant with carboxy-modified substrates. The data are given as
mean ꢂ SD with nꢂ3. The data for the WT enzyme with several sub-
strates were reported previously and are included for comparison.[3,9b,10]
WT
Kd [mm]
S244D
Kd [mm]
Substrate
% HS
% HS
70
1[3]
2[10]
3[10]
5[10]
6[9b]
7[9b]
8[10]
9
ꢃ95
0.28ꢂ0.01
880ꢂ30
110ꢂ3
5
<5
>95
80
1400ꢂ80
4.8ꢂ0.9
660ꢂ36
197ꢂ7
60
10
15
80
40
20
5
30
15
1200ꢂ120
3000ꢂ80
210ꢂ4
[a]
0
0
75
55
–
1300ꢂ79
1500ꢂ40
1400ꢂ80
170ꢂ8
5900ꢂ230
6000ꢂ180
10
14
16
ꢃ2000[b]
[a]
0
–
–
440ꢂ26
310ꢂ10
[a]
Type II
Type II
[a] Not determined because of a low spin-state shift or a low response to
the addition of substrate. [b] Estimated because of solubility problems
before the end of the titration.
activity of the enzyme with a particular substrate, for which
tight substrate binding usually leads to a fast substrate oxida-
tion (Table 3, Table S1). For example, WT CYP199A4 has a high
affinity for a range of para-substituted benzoic acids with
which it is highly active.[3,6b,9b] However, its activity is low for
substrates that include methyl 4-methoxyphenylacetate, 4-me-
thoxybenzonitrile and 4-methoxyphenylboronic acid, in line
with the low affinity of the enzyme for these substrates
(Table 3, Table S1).
The standard turnover assays showed relatively high turn-
over numbers up to 650 (Table 1), and there was no evidence
of the deactivation of the P450 during NADH oxidation (Fig-
ure S9). To further investigate the number of turnovers that
could be catalysed by the S244D mutant we assessed the turn-
over of selected substrates using a lower enzyme concentra-
tion and higher levels of substrate and NADH (Experimental
Section). For 4-methoxybenzoic acid, 4-methoxybenzaldehyde
and 4-methoxybenzamide, no substrate remained in the turn-
overs, and the level of product indicated a total conversion to
a single product to result in total turnovers of 20000 (Table 1,
Figure S10). Analysis of the S244D-catalysed oxidation of 4-me-
thoxyacetophenone revealed little remaining substrate but the
levels of product corresponded to 8100 turnovers (Table 1, Fig-
ure S10). Small amounts of substrate remained in the turnover
of both 4-methoxyphenol and 4-methoxy-1-bromobenzene,
and the turnover numbers were calculated as 14700 and
13000, respectively (Tables 1 and 2, Figure S10). The turnovers
of 4-methoxbenzonitrile and 4-methoxybenyl alcohol, which
had low product formation rate activities, showed total turn-
overs of 1100 and 9100, respectively (Tables 1 and 2, Fig-
ure S10). The high turnover numbers show that the CYP199A4
S244D variant is an efficient catalyst for these substrates suita-
ble for optimisation and for the scale-up of monooxygenase
activity.
Such a relationship between affinity and activity is less ap-
parent in S244D. As an example, in spite of the high NADH oxi-
dation and product formation activities of S244D with 4-me-
thoxybenzamide, the substrate induced a smaller spin-state
shift (20%) and bound fourfold more weakly to S244D (Kd =
3000 mm) than to the WT enzyme (Table 3). Although the
S244D turnover of 4-methoxyphenylboronic acid was 20-fold
slower compared to that of 4-methoxybenzamide, the enzyme
exhibited a similar binding affinity for the boronic acid deriva-
tive (Kd =1500 mm, 20% spin-state shift). However, 4-methoxy-
benzaldehyde bound to S244D and WT CYP199A4 with virtual-
ly the same spin-state shift (80%) and binding affinity (Kd =
210 mm; Table S3, Figures S11–S14) but was oxidised far more
effectively by S244D. The most notable improvement in sub-
strate binding to S244D relative to the WT enzyme was ob-
served with 4-methoxyphenol. Although this substrate induced
negligible changes in the spin-state of WT CYP199A4, a 60%
shift was observed for S244D with 4-methoxyphenol (Table 3).
In addition, this substrate showed the highest binding affinity
of the carboxy-modified substrates to the S244D variant (Kd =
110 mm; Figures S11–S14). Phenol and 2- and 3-methoxyphenol
did not induce any substrate binding, which again highlights
the requirement for the para substituent in the S244D mutant.
The addition of 4-methoxyaminobenzene to S244D resulted
in a type II shift, which redshifted the low-spin Soret band at
l=418 nm by ꢁ1 nm (Figures S11 and S13). The substrate was
bound with moderate affinity to the S244D variant with a disso-
Substrate binding studies with the S244D mutant
Given the high oxidation activity of S244D, we investigated the
substrate binding affinity of this enzyme for each substrate
(Table 3, Table S1, Figures S11–S14). In the WT enzyme, there is
generally a clear relationship between the binding affinity and
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ciation constant of 310 mm. The observation of type II binding
suggests the direct, or indirect, coordination of the N atom of
4-methoxyaminobenzene to the heme iron centre.[18] This
could inhibit the commencement of the P450 catalytic cycle,
which provides a rationale for the lack of product formation
observed. Overall, these results indicate that a low substrate
binding affinity does not imply a low oxidation activity in the
S244D variant, but does provide further evidence of the role of
serine 244 in substrate recognition and binding in WT
CYP199A4.
boxylate group of 4-methoxybenzoate would be expected to
reduce the binding affinity of this substrate. Notably, the tight-
est-binding substrate reported for this mutant is 4-methoxy-
phenol in which the H-bond-accepting benzoic acid moiety is
replaced with a H-bond-donating phenol that reverses but
may maintain the alcohol–acid interaction observed in the WT
enzyme. This substrate was oxidised with high activity by the
S244D mutant, and the modified active site can accommodate
a broader range of functional groups. Overall, the data suggest
that this region of the active site retains the majority of the hy-
drophilic interactions with the substrates. The preference of
this pocket in the mutant, in which the carboxylate group usu-
ally resides, for hydrophilic moieties is observed in the oxida-
tion of 4-methoxytoluene for which 4-methoxybenzyl alcohol
was the sole singly oxidised product observed. The major
product that arose from this turnover was the double oxida-
tion product 4-hydroxybenzyl alcohol. However, it is unclear
whether this arises from further oxidation of the 4-methoxy-
benzyl alcohol or 4-methylphenol (which is not observed in
the turnover). Therefore, we cannot say with certainty that the
S244D variant of CYP199A4 only binds 4-methoxytoluene with
the O-containing methoxy substituent in the hydrophilic
pocket and the more hydrophobic methyl group over the
heme iron centre. In addition, in the turnover of 1,4-dimethox-
ybenzene, the presumed initial product 4-methoxyphenol is
not observed but is believed to be further oxidised rapidly to
the hydroquinone, which indicates a preference of this hydro-
philic pocket for the phenol alcohol moiety.
Discussion
Significant improvements were observed in the monooxyge-
nase activity of S244D relative to that of WT CYP199A4 with
non-benzoic acid substrates. This mutation was identified ini-
tially by using in vivo assays that included the oxidative deme-
thylation of 4-methoxynitrobenzene, which could simply be
followed colorimetrically. The majority of the substrates that
yielded oxidation products were demethylated exclusively at
the para-methoxy position, consistent with previous results re-
ported with CYP199A4 and CYP199A2. This suggests that the
positioning of the substrate in the active site of S244D and
S244N is likely reminiscent of the binding of 4-methoxybenzoic
acid to the WT enzyme, in which the methoxy group held clos-
est to the heme iron centre for selective oxidation (Figure 1).
This is most likely because the majority of the interactions of
the substrate benzene ring and methoxy group observed in
the crystal structure of 4-methoxybenzoic acid bound WT
CYP199A4 (PDB: 4DO1) remains intact in the serine 244 mu-
tants. In addition, the carboxy-modified terminus would
remain close to and could potentially interact with arginine 92,
serine 95 and arginine 243 as well as the acidic and neutral
side chains of aspartate 244 and asparagine 244, respectively.
The binding studies on S244D revealed that, unlike the WT
CYP199A4 enzyme, there did not appear to be a clear relation-
ship between the spin-state shift, binding affinity and the oxi-
dation activity. Other reports on mutant forms of P450 en-
zymes have demonstrated that the spin-state equilibrium is
often not a reliable indicator of the activity towards a given
substrate.[16a,19] Additionally, the serine-to-aspartate mutation
appeared to reduce the overall substrate binding affinity of
CYP199A4. For example, the binding affinity of 4-methoxyphe-
nol, the substrate that bound the most tightly to S244D, was
almost 400-fold lower than that of WT CYP199A4 with 4-me-
thoxybenzoic acid. These results imply that the alteration of
serine 244, a key active site residue in substrate binding, af-
fects the substrate binding affinity dramatically. This may be
a direct effect in which substrate enzyme interactions are dis-
rupted by the negative charge of the aspartate residue or
could potentially be a consequence of structural changes in
the active site induced by this mutation.
Although WT CYP199A4 exhibits a low oxidation activity for
substrates in which the carboxy terminus of the benzoic acid
moiety has been modified, the mutation of serine 244 to an as-
partate residue improves the activity of the enzyme towards
other substrates significantly in spite of generally weaker sub-
strate binding. The total turnover numbers obtained with
S244D CYP199A4 and the highly active substrates were high
and exceeded 10000 without the use of a co-factor regenera-
tion system. In addition, the whole-cell oxidation system oxi-
dised up to 4 mm substrates (100% conversion, total turnover
number of >10000 based on a P450 concentration of
0.35 mm).[3] Further optimisation of both the in vitro and in vivo
systems could lead to an improvement in the total turnover
number. The current work demonstrates the potential of
CYP199A4 variants as biocatalysts for the regioselective oxida-
tive demethylation of benzene substrates at the para position
in the presence of functional groups that are very sensitive to
oxidation such as aldehydes. The cleavage of an aryl methyl
ether often requires harsh conditions such as elevated temper-
ature and the presence of Lewis acids, the reactions are unse-
lective and few catalysts are available.[20] The catalysis of selec-
tive O-demethylation reactions that proceed under ambient
conditions is an attractive methodology for the synthetically
useful deprotection of methoxy groups.
The S244D mutation modifies the active site in the vicinity
of the carboxylate group of the bound p-methoxybenzoate in
the crystal structure and replaces the alcohol functional group
of serine with an acidic aspartate residue. Electrostatic repul-
sions between the negatively charged aspartate and the car-
Importantly, selectivity for the para position is maintained if
additional groups are present. In the turnover of 3,4-dimethox-
ybenzaldehyde, the substrate was oxidised exclusively to 3-me-
thoxy-4-hydroxybenzaldehyde. These results are similar to that
of previous studies on the WT enzyme with 3,4-dimethoxyben-
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50 mm Tris, pH 7.4 that contained 50% v/v glycerol. Glycerol was
removed immediately before use by gel filtration with a 5 mL PD-
10 column (GE Healthcare, UK) by eluting with 50 mm Tris, pH 7.4.
zoic acid, which was oxidised exclusively to 3-methoxy-4-hy-
droxybenzoic acid.[3] The production of 3-methoxy-4-hydroxy-
benzaldehyde from 3,4-dimethoxybenzaldehyde under mild
conditions by CYP199A4 demonstrates the potential of the
CYP199A4 system to be incorporated into alternative bio-syn-
thetic pathways for the synthesis of this commercially impor-
tant flavour and fragrance compound. The low rate of oxida-
tion of 3,4-dimethoxybenzaldehyde by S244D compared to
that of 4-methoxybenzaldehyde implies that the additional
methoxy group has a significant negative influence on the ac-
tivity of the enzyme. This is more pronounced than the differ-
ences observed for WT CYP199A4 activities with 3,4-dimethox-
ybenzoic and 4-methoxybenzoic acids and requires further in-
vestigation.[3] The activity of 3-methoxy-4-hydroxybenzalde-
hyde production could be improved by additional protein
engineering experiments.
The concentration of CYP199A4 was calculated using e419
=
119 mmꢀ1 cmꢀ1 as determined previously.[3,12] The mutant forms of
CYP199A4 were prepared by insertion of a gene fragment (gblock,
Integrated DNA Technologies) that contained the relevant mutation
between the NcoI-SalI (For S95 and F182) or SalI-BstBI restriction
sites of CYP199A4 (for S244; see Supporting Information for de-
tails). Other mutations were made using the Stratagene quick-
change mutagenesis kit according to the manufacturer’s protocol
(Supporting Information) or have been prepared previously (P94V,
S95A, R243L and R243T).[12]
Substrate binding: Spin-state determination and binding
titrations
The high-spin heme content was determined using an enzyme
concentration of 1.5–3.3 mm in 50 mm Tris, pH 7.4, with the addi-
tion of small aliquots of substrate (from a 100 mm stock) until the
spectrum did not change. The percentage shift was estimated (to
approximately ꢂ5%) by comparison with a set of spectra generat-
ed from the sum of the appropriate percentages of the spectra of
the substrate-free (>95% low-spin, Soret maximum at l=418 nm)
and camphor-bound (>95% high-spin, Soret maximum at l=
392 nm) forms of WT CYP101A1.
Conclusions
CYP199A4 has been modified by mutating an active site serine
residue to an aspartate to demethylate a wide range of ben-
zene substrates at the para position efficiently and regioselec-
tively. This occurs under mild conditions in the presence of
more oxidatively sensitive functional groups, such as an alde-
hyde, with high activity, conversion and total turnover. This
could have applications in catalytic and selective chemical
transformations such as the oxidation of 3,4-dimethoxybenz-
aldehyde to 3-methoxy-4-hydroxybenzaldehyde.
To determine the dissociation constant, CYP199A4 was diluted to
1.3–5.5 mm using 50 mm Tris, pH 7.4, in 2.5 mL and 0.5–2 mL ali-
quots of the substrate were added with a Hamilton syringe from 1,
10 or 100 mm stock solutions in ethanol or DMSO. The maximum
difference in the Soret peak-to-trough absorbance (DA) was re-
corded between l=700 and 250 nm. Further aliquots of substrate
were added until the peak-to-trough difference of the Soret band
did not change. The dissociation constants Kd were obtained by fit-
ting DA against total substrate concentration [S] to a hyperbolic
function [Eq. (1)]:
Future work will involve the exploration of the oxidative po-
tential of S244D for additional P450 reactions, which include
hydroxylation, and investigations into the regio- and stereose-
lective nature of these reactions. Studies on additional active
site mutants of CYP199A4 would also provide more informa-
tion on the substrate specificity of this enzyme for para-substi-
tuted benzene derivatives. The colorimetric assays reported
here based on the oxidative demethylation of analogues of 4-
methoxynitrobenzene may enable the rapid screening of such
mutants. Together, these studies could lead to the develop-
ment of optimised CYP199A4-based catalysts for oxidation of
substituted benzenes and related compounds.
DAmax ꢄ ½Sꢅ
ð1Þ
DA ¼
Kd ꢄ ½Sꢅ
Activity assays
In vitro NADH oxidation rate assays were performed with mixtures
(1.2 mL) that contained 50 mm Tris, pH 7.4, 0.5 mm CYP199A4, 5 mm
HaPux, 0.5 mm HaPuR and 100 mgmLꢀ1 bovine liver catala-
se.[3,6a,7a,b,9b,12] Oxygen was bubbled through the buffer solution
before addition of the enzyme components and the mixtures equi-
librated at 308C for 2 min. Substrates were added as a 100 mm
stock solution in ethanol or DMSO to a final concentration of
Experimental Section
General
General reagents, organic substrates and N,O-bis(trimethylsilyl)tri-
fluoroacetamide with trimethylsilylchloride (BSTFA+TMSCl, 99:1)
were from Sigma–Aldrich, Acros, TCI or Merck, Australia. Buffer
components, NADH and isopropyl-b-d-thiogalactopyranoside
(IPTG) were from Astral Scientific, Australia. UV/Vis spectra and
spectroscopic activity assays were recorded at (30ꢂ0.5)8C by
using an Agilent CARY-60 or Varian CARY-5000 spectrophotometer.
1 mm. NADH was added to approximately 320 mm (final A340
=
2.00), and the absorbance at l=340 nm was monitored. The rate
of NADH oxidation was calculated using e340 =6.22 mmꢀ1 cmꢀ1. The
total turnover number was determined using assays set up like
those above with the enzyme of the P450 system used in the
same ratios but with 0.1 mm CYP enzyme, 2 mm substrate and
4 mm NADH.
The amount of product generated in the turnovers was deter-
mined as described in the analysis of metabolites section and used
to calculate the product formation rate, turnover number (TON)
and the coupling efficiency (vide infra). For 4-methoxynitroben-
zene, the turnovers were set up as above but the increase in A410
Enzymes and molecular biology
The production and purification of WT CYP199A4 and its variants
and the electron transfer proteins (HaPux and HaPuR) have been
described elsewhere.[3,7b,9b,12] Proteins were stored at ꢀ208C in
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Full Papers
was monitored and the rate was calculated using e410
=
158Cminꢀ1 up to 2208C. The retention times for the trimethylsilyl
derivatives are given in Table S2.
18.3 mmꢀ1 cmꢀ1 [21]
.
The amount of NADH added to the 4-methoxy-
nitrobenzene turnover was also recorded (using A340). As the sub-
strate was present in excess, it was assumed that all the NADH was
consumed if the A410 did not increase any further.
The amount of product generated in the turnovers was quantified
by calibrating different amounts of the product against the internal
standard used (derivatised product standards were prepared and
used if required for analysis).
The E. coli whole-cell oxidation system, which comprised the plas-
mids pETDuetHaPux/HaPuR and pRSFDuetHaPux/CYP199A4, was
used to assess the oxidation of the substrates and has been de-
scribed previously.[3] The plasmids were transformed into compe-
tent BL21(DE3) cells and grown on LB plates that contained ampi-
cillin (100 mgmLꢀ1) and kanamycin (30 mgmLꢀ1; LBamp/kan). A single
colony was inoculated into 500 mL broth (2xYTamp/kan) in a 2 L flask
and grown at 378C overnight with shaking at 110 rpm. Protein ex-
pression was induced by the addition of 100 mm IPTG (from a 0.5m
stock in H2O), the temperature was reduced to 258C and the
shaker speed to 90 rpm. The growth was allowed to continue for
another 24 h before the cell pellet was harvested by centrifugation
and washed in E. coli minimal media (EMM; K2HPO4 (7 g), KH2PO4
(3 g), trisodium citrate (0.5 g), (NH4)2SO4 (1 g), MgSO4 (0.1 g), 20%
glucose (20 mL) and glycerol (1% v/v) per litre).[16b] The cell pellet
Acknowledgements
This work was supported by ARC grant DP140103229 (to J.J.D.V.
and S.G.B.). S.G.B. acknowledges the ARC for a Future Fellowship
(FT140100355). The authors also thank the University of Adelaide
for M. Phil Scholarships (for R.R.C. and I.C.K.L.).
Keywords: biotransformations · enzyme catalysis · enzymes ·
oxidation · protein engineering
was re-suspended in double the volume EMM (ꢁ3 gcell wet weight Lꢀ1
)
[1] a) P. R. O. de Montellano, Chem. Rev. 2010, 110, 932–948; b) The Ubiqui-
tous Roles of Cytochrome P450 Proteins, Vol. 3, 1st ed. (Eds.: A. Sigel, H.
Sigel, R. Sigel) John Wiley & Sons, Chichester, 2007; c) Cytochrome
P450: Structure, Mechanism, and Biochemistry, 4th ed., (Ed.: P. R. Ortiz de
Montellano), Springer International Publishing, Cham, 2015; d) I. G. De-
nisov, T. M. Makris, S. G. Sligar, I. Schlichting, Chem. Rev. 2005, 105,
2253–2277; e) T. L. Poulos, Chem. Rev. 2014, 114, 3919–3962.
[2] a) M. J. Cryle, J. E. Stok, J. J. De Voss, Aust. J. Chem. 2003, 56, 749–762;
b) F. P. Guengerich, Chem. Res. Toxicol. 2001, 14, 611–650; c) E. M. Isin,
F. P. Guengerich, Biochim. Biophys. Acta Gen. Subj. 2007, 1770, 314–329.
[3] S. G. Bell, A. B. Tan, E. O. Johnson, L. L. Wong, Mol. BioSyst. 2009, 6, 206–
214.
and split into 30 mL aliquots in 250 mL flasks. The substrates were
added to the re-suspended cells to a concentration of 2 mm and
the reactions were then shaken at 200 rpm and 308C. Samples
(1 mL) for HPLC analysis were taken after 3 h at which point a fur-
ther 2 mm substrate was added and a second sample was taken
after 18 h. The supernatant was separated from the cells by centri-
fugation at 13000 rpm before analysis.
Analysis of metabolites
[4] J. Rittle, M. T. Green, Science 2010, 330, 933–937.
[5] J. T. Groves, G. A. McClusky, J. Am. Chem. Soc. 1976, 98, 859–861.
[6] a) S. G. Bell, F. Xu, I. Forward, M. Bartlam, Z. Rao, L.-L. Wong, J. Mol. Biol.
2008, 383, 561–574; b) T. Coleman, R. R. Chao, J. B. Bruning, J. De Voss,
S. G. Bell, RSC Adv. 2015, 5, 52007–52018.
[7] a) S. G. Bell, J. H. McMillan, J. A. Yorke, E. Kavanagh, E. O. Johnson, L. L.
Wong, Chem. Commun. 2012, 48, 11692–11694; b) S. G. Bell, F. Xu, E. O.
Johnson, I. M. Forward, M. Bartlam, Z. Rao, L. L. Wong, J. Biol. Inorg.
Chem. 2010, 15, 315–328; c) F. Hannemann, A. Bichet, K. M. Ewen, R.
Bernhardt, Biochim. Biophys. Acta Gen. Subj. 2007, 1770, 330–344.
[8] a) C. J. Whitehouse, S. G. Bell, L. L. Wong, Chem. Soc. Rev. 2012, 41,
1218–1260; b) V. B. Urlacher, M. Girhard, Trends Biotechnol. 2012, 30,
26–36.
After substrate oxidation incubations, 132 mL of the reaction mix-
ture (the supernatant from the whole-cell oxidation system) was
mixed with 2 mL of an internal standard solution (10 mm 9-hydrox-
yfluorene in ethanol). This was mixed with 66 mL of acetonitrile
before analysis by using HPLC. Calibrations were undertaken with
authentic samples of the products. Analytical HPLC was performed
by using an Agilent 1260 Infinity Pump equipped with an auto-in-
jector connected using an Agilent Eclipse Plus C18 column
(250 mmꢁ4.6 mm, 5 mm) or a Jupiter C18 300A column (250ꢁ
4.6 mm, 5 mm). The products were separated using a gradient be-
tween 20–95 or 20–50% acetonitrile in water (0.1% trifluoroacetic
acid) at a flow rate of 1 mLminꢀ1 over 30 min. Samples were moni-
tored at l=240 or 254 nm. The details of the retention times of
each analyte are given in Table S2.
[9] a) S. G. Bell, N. Hoskins, F. Xu, D. Caprotti, Z. Rao, L. L. Wong, Biochem.
Biophys. Res. Commun. 2006, 342, 191–196; b) S. G. Bell, R. Zhou, W.
Yang, A. B. Tan, A. S. Gentleman, L. L. Wong, W. Zhou, Chemistry 2012,
18, 16677–16688.
[10] T. Coleman, R. R. Chao, J. De Voss, S. G. Bell, Biochim. Biophys. Acta Pro-
teins Proteomics 2016, 1864, 667–675.
[11] R. R. Chao, J. J. De Voss, S. G. Bell, RSC Adv. 2016, 6, 55286–55297.
[12] S. G. Bell, W. Yang, A. B. Tan, R. Zhou, E. O. Johnson, A. Zhang, W. Zhou,
Z. Rao, L. L. Wong, Dalton Trans. 2012, 41, 8703–8714.
For GC analysis, 990 mL of the reaction mixture was mixed with
10 mL of an internal standard solution (10 mm 9-hydroxyfluorene in
ethanol), and the mixture was extracted with 400 mL of ethyl ace-
tate. If samples were derivatised, the mixture was extracted three
times with 400 mL of ethyl acetate and the organic extracts were
combined and dried over MgSO4. Solvent was evaporated under
a stream of N2, and the sample was dissolved in 200 mL anhydrous
acetonitrile. Excess (25 mL) BSTFA+TMSCl (99:1) was added, and
the mixture was left for at least 120 min to produce the trimethyl-
silyl ester of the carboxylic acid group and trimethylsilyl ether of
the alcohol, if formed. The reaction mixtures of derivatised or non-
derivatised mixtures were used directly for GC analysis. GC–MS
data were collected by using a Shimadzu GC-17A using a QP5050A
GC-MS detector and a DB-5 MS fused silica column (30 mꢁ
0.25 mm, 0.25 mm). The injector and interface were maintained at
a constant temperature of 250 and 2808C, respectively. The oven
temperature was held at 1008C for 1 min and then increased at
[13] a) T. Furuya, K. Kino, Biosci. Biotechnol. Biochem. 2009, 73, 2796–2799;
b) T. Furuya, K. Kino, ChemSusChem 2009, 2, 645–649; c) T. Furuya, K.
Kino, Appl. Microbiol. Biotechnol. 2010, 85, 1861–1868; d) F. Xu, S. G.
Bell, Y. Peng, E. O. Johnson, M. Bartlam, Z. Rao, L. L. Wong, Proteins
2009, 77, 867–880.
[14] a) S. G. Bell, N. Hoskins, C. J. C. Whitehouse, L. L. Wong in Metal Ions in
Life Sciences, Vol. 3, 1st ed. (Eds.: A. Sigel, H. Sigel, R. Sigel), John Wiley &
Sons, Chichester, 2007, Chap. 14, pp. 437–476; b) M. T. Reetz, J. Am.
Chem. Soc. 2013, 135, 12480–12496; c) G. D. Roiban, M. T. Reetz, Chem.
Commun. 2015, 51, 2208–2224; d) J. B. Behrendorff, W. Huang, E. M.
Gillam, Biochem. J. 2015, 467, 1–15; e) S. T. Jung, R. Lauchli, F. H. Arnold,
Curr. Opin. Biotechnol. 2011, 22, 809–817; f) R. Fasan, ACS Catal. 2012,
2, 647–666.
[15] T. Furuya, Y. Shitashima, K. Kino, J. Biosci. Bioeng. 2015, 119, 47–51.
ChemCatChem 2016, 8, 1 – 11
www.chemcatchem.org
9
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
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Full Papers
[16] a) S. G. Bell, X. Chen, R. J. Sowden, F. Xu, J. N. Williams, L. L. Wong, Z.
Rao, J. Am. Chem. Soc. 2003, 125, 705–714; b) S. G. Bell, C. F. Harford-
Cross, L. L. Wong, Protein Eng. 2001, 14, 797–802; c) F. Xu, S. G. Bell, Z.
Rao, L. L. Wong, Protein Eng. Des. Sel. 2007, 20, 473–480.
[17] T. Furuya, Y. Arai, K. Kino, Appl. Environ. Microbiol. 2012, 78, 6087–6094.
[18] a) J. H. Dawson, L. A. Andersson, M. Sono, J. Biol. Chem. 1982, 257,
3606–3617; b) J. H. Dawson, L. A. Andersson, M. Sono, J. Biol. Chem.
1983, 258, 13637–13645; c) C. R. Jefcoate, Methods Enzymol. 1978, 52,
258–279; d) C. W. Locuson, J. M. Hutzler, T. S. Tracy, Drug Metab. Dispos.
2007, 35, 614–622.
[19] a) A. B. Carmichael, L. L. Wong, Eur. J. Biochem. 2001, 268, 3117–3125;
b) A. M. Colthart, D. R. Tietz, Y. Ni, J. L. Friedman, M. Dang, T. C. Pochap-
sky, Sci. Rep. 2016, 6, 22035.
[20] a) P. G. M. Wuts, T. W. Greene, Greene’s Protective Groups in Organic Syn-
thesis, 4th ed., John Wiley & Sons, New York, 2007; b) M. Arisawa, Y.
Nihei, T. Suzuki, M. Yamaguchi, Org. Lett. 2012, 14, 855–857.
[21] I. Sakharov, I. E. Makarova, G. A. Ermolin, Comp. Biochem. Physiol. Part B
1988, 90, 709–714.
Received: August 3, 2016
Published online on && &&, 0000
&
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ÝÝ These are not the final page numbers!

FULL PAPERS
Mutant ninja catalysts: The cyto-
R. R. Chao, I. C.-K. Lau, J. J. De Voss,*
S. G. Bell*
chrome P450 enzyme, CYP199A4 from
the bacterium Rhodopseudomonas pal-
ustris HaA2, is modified to expand its
substrate range beyond benzoic acids.
The S244D mutant is able to act on
a range of para-substituted benzenes
with high activity and total turnover to
result in the selective catalytic oxidative
demethylation under mild conditions.
&& – &&
Modification of an Enzyme Biocatalyst
for the Efficient and Selective
Oxidative Demethylation of para-
Substituted Benzene Derivatives
ChemCatChem 2016, 8, 1 – 11
www.chemcatchem.org
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ