A Single-Site Mutation at Ser146 Expands the Reactivity of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Article abstract of DOI:10.1021/acschembio.6b00402

The oxygenase component (C₂) of p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate 3-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate, similar to the reaction of C₂ with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant (k_OH) for 4-APA of 0.028 s^-1 compared to 17 s^-1 for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate constants for both substrates are very similar (2.6 s^-1 for 4-HPA versus 2.5 s^-1 for 4-APA). These data indicate that Ser146 is a key catalytic residue involved in optimizing C₂ reactivity toward a phenolic compound. Removing this hydroxyl group expands C₂ activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-component flavin-dependent monooxygenases that have this conserved Ser residue.

Full text of DOI:10.1021/acschembio.6b00402

Articles
pubs.acs.org/acschemicalbiology
A Single-Site Mutation at Ser146 Expands the Reactivity of the
Oxygenase Component of p‑Hydroxyphenylacetate 3‑Hydroxylase
†
†,‡
,†
§
†
Taweesak Dhammaraj, Chatchadaporn Pinthong, Surawit Visitsatthawong, Chanakan Tongsook,
§
Panida Surawatanawong, and Pimchai Chaiyen*
†Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University,
Rama 6 Road, Bangkok 10400, Thailand
‡
Institute for Innovative Learning, Mahidol University, Nakhon Pathom 73170, Thailand
§
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Rama 6
Road, Bangkok 10400, Thailand
*
S Supporting Information
ABSTRACT: The oxygenase component (C ) of p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter
2
baumannii catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the
phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in
catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better
at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate
3
-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-
aminophenylacetate, similar to the reaction of C with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to
2
the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow
results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant
−
1
−1
(
kOH) for 4-APA of 0.028 s compared to 17 s for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate
−1
−1
constants for both substrates are very similar (2.6 s for 4-HPA versus 2.5 s for 4-APA). These data indicate that Ser146 is a
key catalytic residue involved in optimizing C reactivity toward a phenolic compound. Removing this hydroxyl group expands C
2
2
activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-
component flavin-dependent monooxygenases that have this conserved Ser residue.
1
,2
lavin-monooxygenases are important biocatalysts. These
Most of the flavin-dependent monooxygenases that can
hydroxylate aromatic compounds require the presence of a
phenolic group on the substrate to facilitate the electrophilic
F
enzymes can catalyze the regio-specific incorporation of a
single oxygen atom in the form of a hydroxyl or epoxide group
into a wide range of substrates. Examples of enzymes in this
class that are useful in biocatalysis include Baeyer−Villiger
monooxygenases, styrene monooxygenase, and various aro-
5
,9,10
aromatic substitution reaction.
compounds, a few enzymes can hydroxylate aniline compounds
such as the FADH -utilizing monooxygenase (GTNG_3160)
In addition to phenolic
2
3
−5
matic hydroxylases.
As biocatalysts are instrumental for the
encoded in the anthranilate degradation gene cluster in
Geobacillus thermodenitrificans NG80-2 which catalyzes the
conversion of anthranilate (or 2-aminobenzoate) into 3-
development of green and sustainable manufacturing processes,
the engineering of biocatalysts to broaden their repertoire of
natural enzymatic activities has the potential to provide a
comprehensive enzymatic toolbox to fit a variety of
applications. The expansion of enzymatic specificity to
encompass reactivity toward non-native substrates is generally
accomplished by engineering enzymes through a random,
11
hydroxyanthranilate. Some hydroxylases that can use aniline
derivatives such as 4-aminophenylalkanoic acid or 4-amino-
benzoic acid as substrates have combined activities of
6
1
2−14
hydroxylation with either deamination or decarboxylation.
15
p-Hydroxybenzoate hydroxylase from P. fluorescens and P.
aeruginosa can catalyze ortho-hydroxylation of aniline
1
6
6
−8
semirandom, or rational approach.
The rational engineering
approach, which is the most cost-effective method, requires an
understanding of the catalytic reaction mechanism and of how
the reaction reactivity and specificity is controlled by active site
residues.
Received: May 9, 2016
Accepted: August 19, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acschembio.6b00402
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
derivatives (p-aminobenzoate) but with much smaller hydrox-
ylation rate constants (more than 10-fold) compared to those
for the native phenolic substrates. The hydroxylation of 4-
aminophenylalkanoic acid to produce the corresponding 3-
hydroxy-4-aminophenylalkanoic acid is potentially useful in
biocatalysis because many of these compounds, such as 3-
hydroxy-4-aminophenylacetic acid (3-OH-4-APA), are precur-
sors for the synthesis of pharmacologically active com-
from the coupled enzymes (if multiple turnover reactions were
employed). Under these conditions, the enzyme only catalyzed
one catalytic cycle. Because the complex of C and reduced
2
−
FMN (C :FMNH ) was the limiting reagent, it was oxidized in
2
the excess of oxygen and could not be regenerated in the
absence of reducing agent. The results indicated that 4-
mercaptophenylacetate, 4-methoxyphenylacetate, 4-nitropheny-
lacetate, 4-fluorophenylacetate, and 4-chlorophenylacetate
could not be used as substrates (data not shown) and only 4-
APA could be used (Table 1). HPLC/DAD/MSD chromato-
1
7−22
pounds.
p-Hydroxyphenylacetate 3-hydroxylase (HPAH) from Acine-
tobactor baumannii makes a good candidate starting enzyme for
exploratory engineering to produce an enzyme that can prepare
Table 1. Product Formation from Single Turnover
a
3
-hydroxy-4-aminophenylacetate (3-OH-4-APA) from 4-ami-
Reactions of Wild-Type C , Ser146, and His120 Variants
2
nophenylacetate (4-APA). Native HPAH catalyzes ortho-
with 4-APA (0.2 mM) at pH 6.0−9.0
hydroxylation of p-hydroxyphenylacetate (4-HPA) to form
3
,4-dihydroxyphenylacetate (3,4-DHPA). The enzyme is a two-
component flavin-dependent monooxygenase that consists of
reductase (C ) and oxygenase (C ) components.
2
3,24
The
1
2
reaction mechanism of C is one of the best understood among
2
25−32
the two-component flavin-dependent monooxygenases,
and X-ray crystal structures are available. In addition to 4-
b
33
C
enzyme
pH
% yield of 3-OH-4-APA
2
wild-type
6
7
40 ± 1
31 ± 5
HPA, C can also catalyze the ortho-hydroxylation of other
2
phenolic acids with R groups such as −COOH, −CH COOH,
2
34
8, 9
6
−
CH CH COOH, and trans-CHCHCOOH. C has many
2
2
2
S146A
100.0 ± 0.1
95 ± 3
77 ± 2
66 ± 5
28.7 ± 0.8
9 ± 3
properties that make it suitable for biocatalytic applications
including having a high yield of overexpression and exhibiting
7
28
8
good stability over a wide-range of pH conditions (pH 6−10)
34
9
and temperatures (25−40 °C for more than 24 h). Therefore,
S146C
6
expansion of the activity of C to go beyond hydroxylation of
2
7
phenolic acids will make this flavin-monooxygenase a versatile
enzyme useful for inclusion in a biocatalytic toolbox for
multiple applications.
9
H120N
6
17.1 ± 0.1
7
, 9
In this report, we found that engineering of C at Ser146
2
H120D
6, 7, 9
6, 7, 9
6, 7, 9
6, 9
yielded the S146A variant that is much more effective than the
wild-type enzyme in catalyzing ortho-hydroxylation of 4-
aminophenylacetic acid (4-APA) to generate 3-hydroxy-4-
aminophenylacetic acid (3-OH-4-APA). The kinetics of ligand
binding to wild-type and S146A toward 4-APA and 4-HPA
were shown to be different. Measurement of the hydroxylation
rate constants of the wild-type and S146A enzymes revealed
that, while the wild-type enzyme has a much greater reactivity
toward 4-HPA, the reactivity of S146A toward 4-APA and 4-
HPA are not different. Overall, we have shown that a single-site
H120E
H120K
control (no enzyme)
a
Under this experimental condition, the enzyme could only perform
one catalytic cycle because C :FMNH (limiting reagent) was all
−
2
oxidized in the excess of oxygen. With no excess reducing agent,
−
FMNH could not be regenerated. The reactions were performed by
−
mixing a 250 μL solution of C (200 μM):FMNH (100 μM) in 10
2
mM NaH PO at pH 7.0 with a 250 μL solution of 400 μM substrate
2
4
in various aerobic buffers including 100 mM NaH PO at pH 6.0 and
2
4
mutation at S146 can expand the activity of C to go beyond
2
pH 7.0, 100 mM Tris-HCl at pH 8.0, and 100 mM Gly-NaOH at pH
9.0. The reactions were left at RT for 15 min and then quenched with
500 μL of 0.15 M HCl. The quenched solutions were filtered through
Microcon centrifugal units (10 kDa cutoff), and the filtrates obtained
were analyzed using HPLC/DAD/MSD. The % yield of product was
calculated from the amount of substrate consumed compared to the
the natural phenolic acid substrate to include an aniline
compound.
b
RESULTS AND DISCUSSION
■
Rational engineering of C was performed in order to obtain a
2
limiting agent (reduced flavin) in the reaction.
variant that is superior to the wild-type enzyme in catalyzing
hydroxylation of 4-APA. Transient kinetics (stopped-flow and
rapid-quench flow techniques) indicated that the hydroxyl
grams of the wild-type C reaction with 4-APA are shown in
2
group of Ser146 is key to specifying C reactivity toward a
Supporting Information Figures S2−S4. The peak for the
product as detected by HPLC/MSD-positive mode at 12.88
min showed an m/z of 168 (Supporting Information Figure
S2d), and the peak for the substrate at 15.12 min showed an m/
z of 152 (Supporting Information Figure S2c). The data
indicate that the product has an m/z value of 16 AUs greater
than the substrate (m/z 152), suggesting that the product is the
hydroxylated 4-APA. This product has the same HPLC and
mass spec profiles as the standard 3-hydroxy-4-aminophenyl-
acetic acid (3-OH-4-APA; Supporting Information Figure S5),
2
phenolic compound. Hydroxylation of 4-APA by wild-type and
S146A is more efficient at lower pH. Density functional theory
calculations of the binding energy indicate that ligand binding is
more favorable when 4-APA or His120 is protonated.
Single Turnover Reactions of Wild-type Enzyme with
-APA at pH 7.0. Single turnover reactions of wild-type C2
4
with 4-APA and other aromatic compounds with different types
of substituents were carried out at pH 7.0 (see Methods). We
chose to use single turnover reactions to identify the
hydroxylation product in order to avoid complicating factors
indicating that the wild-type C catalyzes ortho-hydroxylation of
2
B
DOI: 10.1021/acschembio.6b00402
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
-APA to form 3-OH-4-APA (product identity was also
confirmed by NMR, discussed below). Table 1 indicates that
only a 31 ± 5% yield of 3-OH-4-APA was generated from the
single turnover reaction at pH 7.0. The control reaction in the
Articles
4
enzyme (∼31 ± 5%). The chromatographic and MSD
characteristics of the product from the S146A reaction with
4-APA are the same as those of the product from the wild-type
enzyme (Supporting Information Figure S4a−d).
Reactions of Wild-type C and His120 and Ser146
Variants with 4-APA at Various pH’s. Single turnover
absence of C did not show a peak at 12.88 min (Supporting
2
2
Information Figure S3a and d).
Reactions of His120 and Ser146 Variants with 4-APA.
The results of the single turnover reaction above indicate that
reactions of wild-type C , S146A, S146C, H120E, H120K, and
2
H120N with 4-APA (0.2 mM) were carried out over a pH
range of 6.0 to 9.0 (see Methods), and the amount of product
formed by each variant is shown in Table 1. H120D, H120E,
and H120K were not able to catalyze the hydroxylation of 4-
APA at any of these pH values, while the others were more
effective at the lower pH values. For most enzymes, the
hydroxylation yield of 4-APA is greater at lower pH’s, and they
cannot catalyze hydroxylation of 4-APA above pH 7.0. H120N
wild-type C is not an efficient biocatalyst for the hydroxylation
2
of 4-APA. To improve the ability of C to hydroxylate 4-APA,
2
the residues located close to the binding site of the phenolic
moiety were subjected to enzyme engineering. On the basis of
−
the X-ray structure of the C :FMNH :4-HPA complex (PDB
2
3
3
code 2JBT), the hydroxyl group of 4-HPA is located close to
His120 and Ser1462.8 Å (Figure 1a). A previous report
30
hydroxylates 4-APA only at pH 6.0. Wild-type C and S146C
2
hydroxylates 4-APA only at pH 6.0 and 7.0. S146A, the enzyme
with the highest efficiency of 4-APA hydroxylation, was able to
catalyze the hydroxylation of 4-APA over a wide pH range
(
6.0−9.0). Based on these results, S146A was chosen for further
investigations by transient kinetics to compare with the wild-
type C2.
Multiple Turnover Reactions of Wild-Type C2 and
S146A with 4-APA at pH 6.0. Multiple turnover reactions of
wild-type C and S146A with 4-APA at pH 6.0 (Supporting
2
Information Scheme S1a) were carried out to investigate the
bioconversion of 4-APA by these enzymes on a larger scale.
The results show that both enzymes first catalyze hydroxylation
of 4-APA to form 3-OH-4-APA. The 3-OH-4-APA was then
hydroxylated to form 3,5-dihydroxy-4-aminophenylacetic acid
(
3,5-diOH-4-APA; Supporting Information Figure S6, Tables
S1 and S2, and Scheme S1b). Structures of the single and
1
13
−
double hydroxylated products were identified using H and C
NMR spectroscopy (Supporting Information Figures S7−S10).
Results of the multiple turnover reactions also indicate that the
reaction of S146A with 4-APA or 3-OH-4-APA proceeds much
faster than the reaction of the wild-type enzyme. The abilities of
wild type and S146A to catalyze double hydroxylation of 4-APA
Figure 1. (a) Active site structure of the C :FMNH :4-HPA complex
(
2
33
PDB code 2JBT). The optimized geometries of N-protonated 4-
APA and neutral His120 (b) and neutral 4-APA and protonated
His120 (c) in the active site model in the form of C -hydro-
̈
peroxyflavin. Bond distances are shown in Ångstroms.
4a
is similar to the reactions of wild-type C and Y398S with 4-
HPA and p-coumaric acid, in which the double hydroxylated
product is the final product.
2
demonstrated that His120 and Ser146 are important for the
hydroxylation of 4-HPA. His120 in particular may help in
stabilizing the negative charge of the phenolate anion during
the electrophilic aromatic substitution reaction. Mutation of
Ser146 to Ala or Cys decreased the hydroxylation yield of 4-
HPA to ∼60−70% compared to the ∼100% yield of the wild-
type enzyme. As 4-APA has an amino group in place of the
hydroxyl group of 4-HPA, we thought that production of
variants with substitutions at His120 or Ser146, which are
residues close to the phenolic binding site, might provide
enzymes that are superior to the wild-type enzyme in the
hydroxylation of 4-APA.
34
Transient Kinetic Analysis of Wild-Type C and S146A
2
Reactions to Investigate the Determining Factors that
Make S146A More Efficient than the Wild-Type Enzyme
in 4-APA Hydroxylation. The results in the sections above
indicate that S146A is more efficient than the wild-type enzyme
in the hydroxylation of 4-APA, especially at higher pH’s (Table
1
). We carried out transient kinetic experiments to test what
catalytic steps in the reaction were affected by the substitution.
−
A. Reaction of Wild-Type C and S146A:FMNH with
2
Oxygen in the Absence of Substrate at pH 6.0 and pH 9.0
Several His120 and Ser146 variants with substitutions with
negatively charged (H120D and H120E), positively charged
Observed by Stopped-Flow Spectrophotometry. In the
−
absence of substrate, the reactions of C :FMNH or
(
H120K), and neutral (H120N, S146C, S146A) residues were
2
−
S146A:FMNH with oxygen can only result in the formation
explored for their ability to perform 4-APA hydroxylation.
Single turnover reactions of these His120 and Ser146 variants
with 4-APA at pH 7.0 were carried out, and the results are
shown in Table 1. The H120N, H120D, H120E, and H120K
variants could not hydroxylate 4-APA, while S146C was able to
hydroxylate 4-APA with a low product yield of 9 ± 3%
hydroxylation. Among all of the variants explored, S146A was
the most effective enzyme in catalyzing hydroxylation, resulting
in a yield (∼95 ± 3% yield) greater than that of the wild-type
of C -hydroperoxyFMN and the subsequent elimination of
4a
28,30
H
O
2
to give oxidized FMN (Scheme 1a).
The rate
2
constants of C4a-hydroperoxy-FMN formation were calculated
from the first phase of the absorbance increase at 390 nm and at
446 nm, which indicate the formation of oxidized flavin (blue
kinetic traces in Figures 2a,b and 3a,b, Supporting Figures
S11a,b and S12a,b; Table 2). Overall, the rate constants
associated with the two steps in both enzymes (Scheme 1a and
C
DOI: 10.1021/acschembio.6b00402
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Scheme 1. (a) Reaction of Wild-Type C or S146A with Oxygen in the Absence of 4-APA at pH 6.0 or 9.0, (b) Reaction of Wild-
2
Type C with Oxygen in the Presence of 4-APA at pH 6.0 or the Reaction of S146A with Oxygen in the Presence of 4-APA at pH
2
a
b
9
.0, (c) Reaction of S146A with Oxygen in the Presence of 4-APA at pH 6.0
a
The rate constant of step V is thought to be faster than the hydroxylation rate (kOH) because the last phase of the stopped-flow results (flavin
b
oxidation) occurred concomitantly with product formation (hydroxylation, rapid-quench flow results; Figure 2a and Supporting Figure S12a). Rate
constants are described as kOOH = oxygen reaction, kH2O2 = H O elimination, and k = hydroxylation.
2
2
OH
−
Table 2) agree well with the results reported in previous
C. Reaction of Wild-Type C :FMNH with Oxygen in the
2
2
8,30
studies.
Presence of 4-APA at pH 6.0 Observed by Stopped-Flow
Spectrophotometry. In contrast to the reaction of the wild-
type C and 4-APA at pH 9.0, the presence of 4-APA at pH 6.0
−
B. Reaction of Wild-Type C :FMNH with Oxygen in the
2
Presence of 4-APA at pH 9.0 Observed by Stopped-Flow
2
increases the observed rate constant of the second phase of the
reaction to be much faster than the reaction performed in the
Spectrophotometry. As the reaction of the wild-type C with
2
4
1
-APA at pH 9.0 did not give any hydroxylation product (Table
), we performed stopped-flow experiments similar to the
absence of 4-APA (k of 0.039 ± 0.001 s− versus ∼0.0018
1
reox
−
1
s , Figure 2a, black and blue kinetic traces). To address
experiments in section A, but in the presence of 4-APA (2−10
mM) to investigate whether 4-APA can bind to the wild-type
enzyme at this pH. Kinetic traces of the reaction monitored at
−
whether 4-APA can bind to a binary complex of C :FMNH
before the enzyme reacts with oxygen, kinetic traces of the
reaction of C :FMNH (syringe A) mixed with an oxygenated
solution of 4-APA (syringe B) in the stopped-flow instrument
were compared to the reaction of premixed C :FMNH and 4-
APA (syringe A) with oxygenated buffer (syringe B). The
results show that 4-APA does not affect formation of the C -
hydroperoxy-FMN even at 10 mM 4-APA (data not shown),
implying that the ligand binds to the wild-type enzyme only
after the intermediate is formed (Scheme 1b, step II). As the
2
−
2
390 and 446 nm shown in Supporting Figure S11a (black
traces) indicate that the reactions of wild-type enzymes in the
presence of 4-APA even at 10 mM are very similar to the
reaction in the absence of 4-APA (blue traces). These data
suggest that at pH 9.0, 4-APA has essentially no effect on the
reaction of the reduced wild-type enzyme with oxygen,
implying that it cannot bind to the wild-type enzyme.
Therefore, the lack of 4-APA hydroxylation at this pH is likely
due to the lack of 4-APA binding or binding of 4-APA in an
unproductive manner, which does not disrupt the kinetics of
C -hydroperoxyflavin formation and decay.
−
2
4
a
wild-type C can hydroxylate 4-APA (2 mM) at pH 6.0 with
∼83 ± 0% yield (Table 2), the data indicate that the second
phase observed in Figure 2a is comprised of multiple steps
2
including binding of 4-APA to C -hydroperoxyflavin, the
4
a
4a
D
DOI: 10.1021/acschembio.6b00402
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Figure 3. (a) Transient kinetics of the reaction of S146A with oxygen
Figure 2. (a) Transient kinetics of the reaction of wild-type C with
2
in the presence of 4-APA at pH 6.0 (black kinetic traces). A solution of
oxygen in the presence of 4-APA at pH 6.0. A solution of wild-type C2
−
S146A (110 μM):FMNH (50 μM) premixed with 4-APA (2 mM)
−
(
110 μM):FMNH (50 μM) premixed with 4-APA (2 mM) was
was mixed with solutions of 0.26, 0.62, 1.22, and 2.06 mM oxygen and
mixed with solutions of 0.26, 0.62, 1.22, and 2.06 mM oxygen and 4-
APA 2 mM (before-mixing concentrations) in 100 mM NaH PO at
4
-APA 2 mM in 100 mM NaH PO at pH 6.0. Concentrations are as
2 4
2
4
before mixing. Reactions were monitored at 390 and 446 nm using the
stopped-flow spectrophotometer. A plot of peak areas (DAD 220 nm)
of the hydroxylated product 3-OH-4-APA obtained from the rapid-
quench flow experiments under the same conditions is shown by the
red filled circles. A single exponential fit of the data is shown by the red
pH 6.0 at 4 °C (black kinetic traces). Reactions were monitored at 390
and 446 nm using a stopped-flow spectrophotometer. A plot of peak
areas (SIM-168, TIC) of the hydroxylated product 3-OH-4-APA
obtained from the rapid-quench flow experiments under the same
conditions is shown in red filled circles. A single exponential fit of the
data is shown by the red solid line, consistent with an observed
line, consistent with an observed hydroxylation rate constant (kOH) of
−1
2
.5 ± 0.2 s . For reference, kinetics of the same reaction without 4-
−1
hydroxylation rate constant (kOH) of 0.034 s . For reference, kinetics
of the same reaction without 4-APA at pH 6.0 are shown by the blue
traces. (b) A plot of the observed rate constants of the first phase
APA at pH 6.0 are shown in blue. (b) A plot of the observed rate
constants of the first phase (kobs1) versus oxygen concentrations. A
bimolecular constant (kOOH) from this plot was determined as 2.9 ×
(
kobs1) versus oxygen concentration. A bimolecular constant (kOOH)
5
−1 −1
1
0 M s .
6
−1 −1
from this plot was determined as 1.1 × 10 M s .
first kinetic phase (black versus blue traces, Supporting Figure
S12a, Table 2). These results indicate that 4-APA can bind to
S146A only after C -hydroperoxyflavin formation (Scheme 1b,
uncoupling reaction to eliminate H O , the coupling reaction to
2
2
form the hydroxylated product, and dehydration to form the
oxidized enzyme (Scheme 1b, steps II, III, IV, and V).
D. Measurement of the Hydroxylation Rate Constant of
4a
step II). The second observed phase was comprised of multiple
steps, including the binding of 4-APA to C -hydroperoxyflavin,
4
a
the Reaction of Wild-Type C and 4-APA at pH 6.0 Using
2
1
the uncoupling reaction to eliminate H O with k
= 0.24 s−
Rapid-Quench Flow Techniques. In order to directly measure
the rate constant associated with the hydroxylation of 4-APA,
rapid-quench experiments were carried out to measure the
amount of 3-OH-4-APA product formed over time. The
observed rate constant of hydroxylation (kOH) was determined
2
2
2 2
H O
(Table 2), the coupling reaction to form the hydroxylated
product with kOH = 1.07 s− (Table 2), and the dehydration
reaction to yield the oxidized enzyme (Scheme 1b, steps II, III,
1
IV, and V).
−1
−
as ∼0.034 ± 0.004 s (Figure 2a, red filled circles and red
line). Because the reaction undergoes bifurcation pathways of
coupling and uncoupling reactions (Scheme 1b), the observed
hydroxylation rate constant is the summation of the rate
constants from both pathways as represented by eq 1. Based on
a product ratio of 83% (Table 2), the actual rate constant of
hydroxylation (kOH) and H O elimination (k ) under these
F. Reaction of S146A:FMNH with O
2
in the Presence of 4-
APA at pH 6.0 Monitored by Stopped-Flow Spectropho-
−
tometry. The kinetics of the reaction of S146A:FMNH with
O in the presence of 4-APA at pH 6.0 resulted in four phases
2
(Figure 3a, black traces). Unlike the reaction of S146A at pH
9.0, the first and second phases are dependent on oxygen,
implying that these two phases are reactions of enzyme-bound
reduced flavin with oxygen to form C -hydroperoxy-FMN. The
2
2
2 2
H O
−1
−1
4a
conditions can be calculated as 0.028 s and 0.006 s ,
respectively (Table 2).
first phase occurred during dead-time0.01 s. During this
phase, the absorption at 390 nm increased from ∼0.04−0.05
AU without any significant change in absorption at 446 nm. A
plot of the observed rate constants of this phase (kobs1) versus
oxygen concentration (Figure 3b) yielded a calculated
ratio of product = k
=
kOH
OH
kobs
kOH + k
H O
(1)
2
2
−
5
−1 −1
E. Reaction of S146A:FMNH with O in the Presence of 4-
bimolecular rate constant (kOOH) of 2.9 × 10 M s . This
2
APA at pH 9.0 Observed by Stopped-Flow Spectropho-
value is similar to the bimolecular rate constant of the reaction
5
−1 −1
tometry. Unlike wild-type C at pH 9.0 (section B), S146A can
without 4-APA (2.8 × 10 M s ; Figure 3a, blue traces;
Table 2), indicating that this phase is the reaction of
S146A:FMNH− with oxygen to form the S146A:C -hydro-
2
bind 4-APA at this pH. The presence of 4-APA increased kreox
of the second phase of the S146A reaction with oxygen from
4
a
1
−1
0
.028 ± 0.001 s− to ∼1.64 ± 0.01 s but did not affect the
peroxy-FMN intermediate (step I in Scheme 1c). The second
E
DOI: 10.1021/acschembio.6b00402
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Table 2. Kinetic Rate Constants of Reactions of Wild-Type and S146A at pH 6.0 and 9.0 According the Scheme 1a−c
a
kOOH (M−1 s−1
b
kobs (s−1
c
kH2O2 (× 10−3 s−1
d
kOH (× 10−3 s−1
e
reaction
substrate
4-APA
pH
% yield of 3-OH-4-APA
)
)
)
)
wild-type C2
6
9
6
9
6
9
6
9
83 ± 1
1.1 × 106
1.5 × 10
1.1 × 106
1.5 × 10
2.9 × 105
6
28
6
155 (±1)
1.8 (±0.1)
159 (±1)
wild-type C2
S146A
6
4-APA
100 ± 1
82 ± 3
26.2, 32.2, 38.6, 41.4
2500 (±200)
1070
5
2.5 × 10
240
S146A
2.9 × 105
2.1 × 105
6.8 (±0.2)
28 (±1)
a
%
Yield of product with 2 mM 4-APA substrate was calculated based on HPLC chromatograms of the formed product and values from a standard 3-
b
c
OH-4-APA. k
= bimolecular rate constant of C :FMNH reacting with oxygen (step I in Scheme 1a,b,c) k
= observed rate constants of
obs2
OOH
2
−
−1
S146A:FMNH :4APA reacting with 0.13, 0.31, 0.61, and 1.03 mM oxygen at pH 6.0 were determined as 26.2, 32.2, 38.6, and 41.4 s , respectively
d
e
(Scheme 1c, step III). k
= rate constant of hydrogen peroxide elimination (Scheme 1a, step II; Scheme 1b, step III) k = 4-APA hydroxylation
H
2
O
2
O
H
rate constant (Scheme 1b, step IV; Scheme 1c, step V)
Table 3. Comparison of Mode of Binding and Hydroxylation Rate Constant (kOH) of Wild-Type and S146A Reactions with 4-
HPA and 4-APA at pH 6.0
wild-type C2
mode of binding
binds to C :FMNH− and C -hydroperoxyFMN
S146A
mode of binding
binds to C :FMNH− and C -hydroperoxyFMN
substrate
kOH (s−1)
kOH (s−1
)
a
b
c
4
-HPA
-APA
17 (pH 6−10)
2.6 (pH 7, 9)
2.5 (pH 6.0)
2
4a
2
4a
4
only binds to C -hydroperoxyFMN
0.028 (pH 6.0)
30 c
binds to C :FMNH− and C -hydroperoxyFMN
4
a
2 4a
a
28
b
Data are from Ruangchan et al. Data are from Tongsook et al. The hydroxylation rate constant and product yield of wild-type C with 4-HPA
2
28
30
are independent of pH (pH 6.0−10.0). As the product yield of S146A and 4-HPA is also constant throughout a pH range of 6.0−9.0 and the
hydroxylation rate constants at pH 7.0 and 9.0 are the same, it is likely that the hydroxylation rate constant of S146A and 4-HPA at pH 6.0 is also 2.6
−
1
s .
phase occurred during 0.01−0.1 s and was oxygen dependent,
giving an absorption increase at 390 nm of ∼0.04−0.09 AU
with no significant change in absorbance at 446 nm. The
observed rate constants (kobs2) of this phase in the presence of
independent of oxygen concentration. These phases involve
hydroxylation and the dehydration of C -hydroxyFMN to
4a
return to the oxidized FMN (Scheme 1c, steps V and VI).
G. Measurement of the Hydroxylation Rate Constant of
the Reaction of S146A and 4-APA at pH 6.0 Using Rapid-
Quench Flow Techniques. The observed hydroxylation rate
constant kOH measured using rapid-quench flow techniques
(Figure 3a, red filled circles and red line) indicates that the
hydroxylation step occurs during ∼0.05−2 s, essentially
concomitant with the third phase of the stopped-flow traces
(Figure 3a, black traces), which corresponds to a hydroxylation
rate constant of 2.5 ± 0.2 s− (Figure 3a, red filled circles and
red line; Scheme 1c, step V). Because the reaction under these
conditions yields ∼100% of product (Table 2), the observed
rate constant is thus equal to the actual hydroxylation rate
constant (Table 2; Scheme 1c, step V). Interestingly, this
hydroxylation rate constant is similar to the hydroxylation rate
0
3
.13, 0.31, 0.61, and 1.03 mM oxygen were determined as 26.2,
−1
2.2, 38.6, and 41.4 s , respectively (Table 2). As at pH 6.0, a
−
ternary complex of S146A:FMNH :4-APA can be formed
before reaction with oxygen (Supporting Information Figure
S13a,b), the second phase in Figure 3a is likely a bimolecular
−
reaction of the S146A:FMNH :4-APA complex with oxygen to
form the S146A:C -hydroperoxy-FMN:4-APA complex
4
a
1
(Scheme 1c, step III). In principle, observed rate constants of
this phase should be linearly dependent on oxygen concen-
tration. However, as their observed rate constant values are not
well separated from the first phase of reaction, accurate
observed rate constants could not be obtained. Therefore, a
bimolecular rate constant (kOOH) for the second phase cannot
be determined. This reaction is similar to the reaction of wild-
30
constant of the reaction of S146A and 4-HPA (Table 3).
2
6,28
type C with 4-HPA,
in that it has two pathways for oxygen
Catalytic Feature that Enables S146A to be Efficient in
Hydroxylation of 4-APA. All of the transient kinetic results
2
−
reaction. In the first pathway, S146A:FMNH can react with
oxygen directly to form S146A:C -hydroperoxyFMN (with a
bimolecular rate constant, kOOH, of 2.9 × 10 M s ) before
indicate that the wild-type C does not react with 4-APA
4
a
2
5
−1 −1
efficiently (Table 3). In contrast, kinetics of the reactions of
S146A with 4-APA and with 4-HPA are similar. Notably, S146A
catalyzes hydroxylation of 4-APA and 4-HPA with similar rate
binding 4-APA to form the S146A:C -hydroperoxyFMN:4-
4
a
APA complex (Scheme 1c, steps I and IV). In the second
−
−1
−1
pathway, S146A:FMNH can prebind with 4-APA to form
constants at pH 6.0 (2.5 s versus 2.6 s , Table 3). These
results indicate that the interaction between Ser146 and the
phenolic group of 4-HPA is a key dictating factor in optimizing
−
S146A:FMNH :4-APA and then react with oxygen to generate
the ternary complex (Scheme 1c, steps II and III). The third
phase occurred at 0.1−1.2 s with an absorption decrease at 390
nm (∼0.015−0.025 AU), corresponding to an observed rate
C reactivity toward 4-HPA. Upon removal of the Ser146
2
hydroxyl group, the enzyme (S146A) loses its ability to
discriminate between 4-APA and 4-HPA and can subsequently
catalyze hydroxylation of the unnatural substrate 4-APA with
similar reactivity as the natural substrate 4-HPA.
−
1
constant (k ) of ∼1.5 ± 0.2 s . The last phase occurred at
obs3
1
.2−80 s with a large absorption increase at 446 nm (∼0.28
AU) concomitant with an absorption decrease at 390 nm. The
observed rate constant of this phase (k or kreox) was
Density Functional Theory Analysis Explains the pH
obs4
−
1
determined as ∼0.048 ± 0.001 s . The last two phases were
Effect on the Reaction of Wild-Type C and 4-APA.
2
F
DOI: 10.1021/acschembio.6b00402
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Results in Table 1 and Figure 2 and 3 indicate a higher
efficiency in hydroxylation and binding for the wild-type and
S146A enzymes at lower pH values. As this effect may be due to
ACKNOWLEDGMENTS
■
This research was financially supported by The Thailand
Research Fund through grants RTA5980001 (to P.C.) and
Royal Golden Jubilee Ph.D. Program Grant PHD/0335/2551
the protonation states of 4-APA (−NH moiety) and active site
2
residues (such as His120), density functional theory (DFT)
was employed to investigate the binding energy of 4-APA to the
(
to T.D.) and RSA5980028 (to P.S.), a grant from Mahidol
35
University (to P.C. T.D., C.P. and C.T.) and the Center of
Excellence for Innovation in Chemistry (PERCH-CIC) (to P.S.
and S.V.).
active site model in the form of C -hydroperoxyflavin under
4
a
three states: (A) protonated 4-APA and neutral His120 (Figure
b), (B) neutral 4-APA and protonated His120 (Figure 1c),
1
and (C) neutral 4-APA and neutral His120 (Supporting
Information Figure S14).
The DFT results indicate that states A and B which contain
an extra proton on 4-APA or His120 have binding energies of
REFERENCES
■
(1) Huijbers, M. M., Montersino, S., Westphal, A. H., Tischler, D.,
and van Berkel, W. J. (2014) Flavin dependent monooxygenases. Arch.
Biochem. Biophys. 544, 2−17.
2) Torres Pazmino, D. E., Winkler, M., Glieder, A., and Fraaije, M.
W. (2010) Monooxygenases as biocatalysts: classification, mechanistic
−54.3 kcal/mol and −47.3 kcal/mol, respectively. These values
(
are greater than those of state C (−37.2 or −39.8 kcal/mol) in
which both moieties are neutral. The greater stabilization in
states A and B is mainly due to the presence of stronger
hydrogen bond interactions than in state C (Figure S14). States
aspects and biotechnological applications. J. Biotechnol. 146, 9−24.
3) Leisch, H., Morley, K., and Lau, P. C. (2011) Baeyer-Villiger
(
monooxygenases: more than just green chemistry. Chem. Rev. 111,
165−4222.
4) Torres Pazmino, D. E., Dudek, H. M., and Fraaije, M. W. (2010)
Baeyer-Villiger monooxygenases: recent advances and future chal-
lenges. Curr. Opin. Chem. Biol. 14, 138−144.
A and B likely represent the protonation state of the C :4-APA
2
4
(
complex at pH 6.0, while state C likely represents the
protonation state at pH 9.0. Therefore, the DFT results
suggest that at lower pH values, the binding energy of 4-APA is
more favorable, allowing the reaction to proceed with higher
efficiency under these conditions.
Conclusion. In conclusion, our work has demonstrated that
it is possible to expand the reactivity of a two-component
flavin-monooxygenase to go beyond the natural substrate. The
variant S146A can catalyze the hydroxylation of an aniline
substrate with similar reactivity as the natural phenolic
substrate. The knowledge acquired by these studies may be
(5) Montersino, S., Tischler, D., Gassner, G. T., and van Berkel, W. J.
(2011) Catalytic and structural features of flavoprotein hydroxylases
and epoxidases. Adv. Synth. Catal. 353, 2301−2319.
(
6) Toogood, H. S., and Scrutton, N. S. (2013) Enzyme engineering
toolbox−a ‘catalyst’for change. Catal. Sci. Technol. 3, 2182−2194.
7) Bornscheuer, U. T., Huisman, G. W., Kazlauskas, R. J., Lutz, S.,
Moore, J. C., and Robins, K. (2012) Engineering the third wave of
biocatalysis. Nature 485, 185−194.
8) Bornscheuer, U. T. (2013) From commercial enzymes to
biocatalysts designed by protein engineering. Synlett 24, 150−156.
9) Fagan, R. L., and Palfey, B. A. (2010) Flavin-Dependent
(
(
applied for future enzyme engineering of C homologues
2
(implications proposed in the Supporting Information).
(
Enzymes, in Comprehensive Natural Products II (Liu, H.-W., and
Mander, L., Ed.), pp 37−113, Elsevier, Oxford.
METHODS
■
(
10) Chaiyen, P. (2010) Flavoenzymes catalyzing oxidative aromatic
All methods used in this research are described in the Supporting
Information.
ring-cleavage reactions. Arch. Biochem. Biophys. 493, 62−70.
(11) Liu, X., Dong, Y., Li, X., Ren, Y., Li, Y., Wang, W., Wang, L., and
Feng, L. (2010) Characterization of the anthranilate degradation
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.6b00402.
■
pathway in Geobacillus thermodenitrificans NG80−2. Microbiology 156,
*
S
5
(
89−595.
12) Mizutani, Y., Narikawa, T., Satoh, T., Sakurai, N., Kaji, H.,
Yamada, S., and Samejima, T. (1999) A New UV method for serum γ-
glutamyltransferase assay using recombinant 4-aminobenzoate hydrox-
ylase as a coupling enzyme. J. Biochem. 126, 347−353.
Methods; HPLC-chromatograms of single turnover
reactions of 4-APA reactions; identification of product
from single turnover reaction of 4-APA by comparison
with a standard 3-OH-4-APA; multiple turnover
(13) Tsuji, H., Oka, T., Kimoto, M., Hong, Y. M., Natori, Y., and
Ogawa, T. (1996) Cloning and sequencing of cDNA encoding 4-
aminobenzoate hydroxylase form Agaricu bisporus. Biochim. Biophys.
Acta, Gene Struct. Expression 1309, 31−36.
reactions of wild-type C and S146A with 4-APA at pH
2
(14) Bando, N., Ogawa, T., Tsuji, H., and Sasaoka, K. (1990)
Enzymatic method for selective determination of 4-aminobenzoic acid
in urine. Clin. Chem. 36, 1937−1940.
1
13
6
.0; H and C NMR spectra of products from multiple
turnover reactions; reactions of wild-type C2 or
−
S146A:FMNH with oxygen in the absence of substrate
(15) Entsch, B., Massey, V., and Claiborne, A. (1987) para-
Hydroxybenzoate hydroxylase containing 6-hydroxy-FAD is an
effective enzyme with modified reaction mechanisms. J. Biol. Chem.
at pH 6.0 and pH 9.0 monitored by stopped-flow
spectrophotometry; reactions of wild-type C and S146A
reactions in the presence of 4-APA at pH 9.0 observed by
stopped-flow spectrophotometry and rapid-quench
technique (PDF)
2
2
(
62, 6060−6068.
16) Gatti, D. L., Entsch, B., Ballou, D. P., and Ludwig, M. L. (1996)
pH-dependent structural changes in the active site of p-hydrox-
ybenzoate hydroxylase point to the importance of proton and water
movements during catalysis. Biochemistry 35, 567−578.
(17) Frank, R., Bahrenberg, G., Christoph, T., Schiene, K., De, V. J.,
Saunders, D. J., Sundermann, B., and Lee, J. (2008) Novel Vanilloid
receptor ligands and the use thereof for the production of
pharmaceuticals. PCT Pat. Appl. WO2008125342 A2, Oct 23, 2008.
AUTHOR INFORMATION
Corresponding Author
Phone: +66-2201 5596. Fax: +66-2354 7174. E-mail: pimchai.
cha@mahidol.ac.th.
Notes
■
*
(18) Muro, F., Iimura, S., Yoneda, Y., Chiba, J., Watanabe, T.,
Setoguchi, M., Takayama, G., Yokoyama, M., Takashi, T., Nakayama,
A., and Machinaga, N. (2009) A novel and potent VLA-4 antagonist
The authors declare no competing financial interest.
G
DOI: 10.1021/acschembio.6b00402
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
based on trans-4-substituted cyclohexanecarboxylic acid. Bioorg. Med.
Chem. 17, 1232−43.
Flavin-Dependent Monooxygenase: A Nearly Barrierless Formation of
C4a-Hydroperoxyflavin via Proton-Coupled Electron Transfer. J. Am.
Chem. Soc. 137, 9363−9374.
(
19) Brittain, D. R., Davies, G. M., Johnstone, C., and Large, M. S.
(
2004) Benzoxazole derivatives for use as integrin inhibitors. PCT Pat.
Appl. EP1144393B1, Feb 11, 2004.
20) Fischer, R., Hasegawa, H., Hessler, G., Komura, H., Lehmann,
(
T., Mizoguchi, M., Mueller, G., Okigami, H., Tajimi, M., and
Ziegelbauer, K. (2001) Piperidyl carboxylic acids as integrin
antagonists. PCT Pat. Appl. WO2001058871A1, Aug 16, 2001.
(
21) Dunwell, D. W., and Evans, D. (1977) Synthesis and
antiinflammatory activity of some 2-aryl-6-benzoxazoleacetic acid
derivatives. J. Med. Chem. 20, 797−801.
(
22) Dawson, W. (1983) Method of treating hypersensitivity disease
with benzoxazole derivatives. U.S. Patent No. 4,416,892 A, Nov 22,
983.
23) Chaiyen, P., Suadee, C., and Wilairat, P. (2001) A novel two-
protein component flavoprotein hydroxylase. Eur. J. Biochem. 268,
550−5561.
24) Thotsaporn, K., Sucharitakul, J., Wongratana, J., Suadee, C., and
Chaiyen, P. (2004) Cloning and expression of p-hydroxyphenylacetate
-hydroxylase from Acinetobacter baumannii: evidence of the
1
(
5
(
3
divergence of enzymes in the class of two-protein component aromatic
hydroxylases. Biochim. Biophys. Acta, Gene Struct. Expression 1680, 60−
6
6.
(
25) Sucharitakul, J., Chaiyen, P., Entsch, B., and Ballou, D. P. (2005)
The reductase of p-hydroxyphenylacetate 3-hydroxylase from
Acinetobacter baumannii requires p-hydroxyphenylacetate for effective
catalysis. Biochemistry 44, 10434−10442.
(
26) Sucharitakul, J., Chaiyen, P., Entsch, B., and Ballou, D. P. (2006)
Kinetic mechanisms of the oxygenase from a two-component enzyme,
p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii.
J. Biol. Chem. 281, 17044−17053.
(
27) Sucharitakul, J., Phongsak, T., Entsch, B., Svasti, J., Chaiyen, P.,
and Ballou, D. P. (2007) Kinetics of a two-component p-
hydroxyphenylacetate hydroxylase explain how reduced flavin is
transferred from the reductase to the oxygenase. Biochemistry 46,
8
(
(
611−8623.
28) Ruangchan, N., Tongsook, C., Sucharitakul, J., and Chaiyen, P.
2011) pH-dependent studies reveal an efficient hydroxylation
mechanism of the oxygenase component of p-hydroxyphenylacetate
-hydroxylase. J. Biol. Chem. 286, 223−233.
29) Thotsaporn, K., Chenprakhon, P., Sucharitakul, J., Mattevi, A.,
and Chaiyen, P. (2011) Stabilization of C -hydroperoxyflavin in a
3
(
4a
two-component flavin-dependent monooxygenase is achieved through
interactions at flavin N5 and C4a atoms. J. Biol. Chem. 286, 28170−
2
8180.
(
30) Tongsook, C., Sucharitakul, J., Thotsaporn, K., and Chaiyen, P.
2011) Interactions with the substrate phenolic group are essential for
hydroxylation by the oxygenase component of p-hydroxyphenylacetate
-hydroxylase. J. Biol. Chem. 286, 44491−44502.
31) Phongsak, T., Sucharitakul, J., Thotsaporn, K., Oonanant, W.,
(
3
(
Yuvaniyama, J., Svasti, J., Ballou, D. P., and Chaiyen, P. (2012) The C-
terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from
Acinetobacter baumannii is an autoinhibitory domain. J. Biol. Chem.
2
(
87, 26213−26222.
32) Sucharitakul, J., Tinikul, R., and Chaiyen, P. (2014) Mechanisms
of reduced flavin transfer in the two-component flavin-dependent
monooxygenases. Arch. Biochem. Biophys. 555, 33−46.
(
33) Alfieri, A., Fersini, F., Ruangchan, N., Prongjit, M., Chaiyen, P.,
and Mattevi, A. (2007) Structure of the monooxygenase component of
a two-component flavoprotein monooxygenase. Proc. Natl. Acad. Sci.
U. S. A. 104, 1177−1182.
(
34) Dhammaraj, T., Phintha, A., Pinthong, C., Medhanavyn, D.,
Tinikul, R., Chenprakhon, P., Sucharitakul, J., Vardhanabhuti, N.,
Jiarpinitnun, C., and Chaiyen, P. (2015) p-Hydroxyphenylacetate 3-
Hydroxylase as a Biocatalyst for the Synthesis of Trihydroxyphenolic
Acids. ACS Catal. 5, 4492−4502.
(
35) Visitsatthawong, S., Chenprakhon, P., Chaiyen, P., and
Surawatanawong, P. (2015) Mechanism of Oxygen Activation in a
H
DOI: 10.1021/acschembio.6b00402
ACS Chem. Biol. XXXX, XXX, XXX−XXX