Regioselective hydroxylation pathway of tenatoprazole to produce human metabolites by Bacillus megaterium CYP102A1

Article abstract of DOI:10.1016/j.procbio.2019.09.014

Tenatoprazole, a proton pump inhibitor drug candidate, is developed as an acid inhibitor to treat gastric acid hypersecretion disorders, such as gastric ulcer and reflux esophagitis. Tenatoprazole is known to be metabolized to three major metabolites—tenatoprazole sulfone, 5’-hydroxylated metabolite, and tenatoprazole sulfide—in human livers mainly by CYP2C19 and CYP3A4. In this study, an enzymatic strategy for the production of human metabolites of tenatoprazole was developed using bacterial P450 enzymes. A set of CYP102A1 mutants catalyzed the regioselective hydroxylation reactions of tenatoprazole. The major product of tenatoprazole by CYP102A1 is 5’-OH tenatoprazole, a major human metabolite produced by human CYP2C19 and CYP3A4. As another major metabolite of tenatoprazole, tenatoprazole sulfide is formed via a non-enzymatic conversion without a P450 system. In addition, 5’-OH tenatoprazole sulfide was found as a minor metabolite, which can be formed via a 5’-hydroxylation reaction of tenatoprazole sulfide by CYP102A1 and/or a non-enzymatic reduction of 5’-OH tenatoprazole to a sulfide form. Chemical synthesis of the 5’-OH tenatoprazole is not currently possible. In conclusion, an enzymatic synthesis of 5’-OH tenatoprazole, a major human metabolite of tenatoprazole, was developed by using mutants of CYP102A1 from Bacillus megaterium as a biocatalyst and tenatoprazole as a substrate.

Full text of DOI:10.1016/j.procbio.2019.09.014

Process Biochemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Regioselective hydroxylation pathway of tenatoprazole to produce human
metabolites by Bacillus megaterium CYP102A1
a
a,b
a
a
a
Thien-Kim Le , Gun-Su Cha , Hyun-Hee Jang , Thi Huong Ha Nguyen , Tiep Thi My Doan ,
c
c
d
d
a,⁎
Young Ju Lee , Ki Deok Park , Yumi Shin , Dong-Hyun Kim , Chul-Ho Yun
b
a
School of Biological Sciences and Technology, Chonnam National University, 77 Yongbongro, Gwangju 61186, Republic of Korea
Namhae Garlic Research Institute, 2465-8 Namhaedaero, Gyeongsangnamdo 52430, Republic of Korea
c
Gwangju Center, Korea Basic Science Institute, Gwangju 61186, Republic of Korea
d
Department of Pharmacology and Pharmacogenomics Center, Inje University of College of Medicine, Busan 47392, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Tenatoprazole, a proton pump inhibitor drug candidate, is developed as an acid inhibitor to treat gastric acid
hypersecretion disorders, such as gastric ulcer and reflux esophagitis. Tenatoprazole is known to be metabolized
to three major metabolites—tenatoprazole sulfone, 5’-hydroxylated metabolite, and tenatoprazole sulfide—in
human livers mainly by CYP2C19 and CYP3A4. In this study, an enzymatic strategy for the production of human
metabolites of tenatoprazole was developed using bacterial P450 enzymes. A set of CYP102A1 mutants catalyzed
the regioselective hydroxylation reactions of tenatoprazole. The major product of tenatoprazole by CYP102A1 is
C-hydroxylation
CYP102A1
Human metabolite
Metabolic pathway
Tenatoprazole
Tenatoprazole sulfide
5
’-OH tenatoprazole, a major human metabolite produced by human CYP2C19 and CYP3A4. As another major
metabolite of tenatoprazole, tenatoprazole sulfide is formed via a non-enzymatic conversion without a P450
system. In addition, 5’-OH tenatoprazole sulfide was found as a minor metabolite, which can be formed via a 5’-
hydroxylation reaction of tenatoprazole sulfide by CYP102A1 and/or a non-enzymatic reduction of 5’-OH te-
natoprazole to a sulfide form. Chemical synthesis of the 5’-OH tenatoprazole is not currently possible. In con-
clusion, an enzymatic synthesis of 5’-OH tenatoprazole, a major human metabolite of tenatoprazole, was de-
veloped by using mutants of CYP102A1 from Bacillus megaterium as a biocatalyst and tenatoprazole as a
substrate.
1
. Introduction
to enable a degree of symptom relief and healing of gastric lesions that
is superior to other PPIs. Thus, it can be effectively used in treating
Tenatoprazole (5-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-
atypical and esophageal symptoms of gastroesophageal reflux, digestive
bleeding, and dyspepsia [1].
yl)methylsulfinyl]-1H-imidazo[4,5-b]pyridine) is a proton pump in-
hibitor (PPI) drug candidate that is undergoing clinical testing as a
potential treatment for reflux esophagitis and peptic ulcers [1]. Tena-
toprazole has an imidazopyridine ring in place of the benzimidazole
moiety found in most other PPIs, such as omeprazole (6-methoxy-2-[(4-
methoxy-3,5-dimethylpyridin-2-yl)methanesulfinyl]-1H-1,3-benzodia-
zole) and lansoprazole (2-[[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-
Major metabolites of tenatoprazole in humans are 5’-OH tenato-
prazole, tenatoprazole sulfone, and tenatoprazole sulfide [5]. CYP2C19
and CYP3A4 are known to oxidize tenatoprazole to 5’-OH tenatoprazole
and tenatoprazole sulfone [6]. Omeprazole is also primarily oxidized in
human livers by CYP2C19 and CYP3A4 to produce 5’-OH omeprazole
and omeprazole sulfone, respectively [7]. Omeprazole sulfide is a major
metabolite of omeprazole [8]. In addition, 5’-OH omeprazole sulfide
was identified in human urine [9]. Previously known information re-
garding omeprazole metabolism suggests that the metabolism of tena-
toprazole may be similar to the metabolic pathway of omeprazole
[9,10].
2
-yl]methylsulfinyl]-1H-benzimidazole) (Fig. 1). Interestingly, it has a
plasma half-life that is about seven times longer than that of other PPIs
[
2]. Tenatoprazole demonstrated better acid suppression than S-ome-
prazole in healthy subjects [3,4]. Furthermore, it was shown that higher
doses of tenatoprazole produced greater acid suppression in a dose-
dependent fashion [3]. As tenatoprazole is endowed with a markedly
prolonged action duration, the clinical data have shown tenatoprazole
The importance of drug metabolites in safety testing (MIST) during
drug development was guided by the Food and Drug Administration in
⁎
Corresponding author.
E-mail address: chyun@jnu.ac.kr (C.-H. Yun).
https://doi.org/10.1016/j.procbio.2019.09.014
Received 26 March 2019; Received in revised form 11 September 2019; Accepted 12 September 2019
1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Thien-Kim Le, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.09.014

T.-K. Le, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
Fig. 1. Chemical structures of common proton pump inhibitors, including tenatoprazole, omeprazole, lansoprazole, and pantoprazole.
2
008, and eventually, broader regulatory agreement was reached with
2. Materials and methods
the release of guidance from the International Conference on
Harmonization (ICH M3(R2); 2009) [11]. During the last decade, major
changes have occurred in experimental methods to identify and quan-
tify metabolites, to evaluate the coverage of metabolites, and to de-
termine the timing of critical clinical and nonclinical studies [12]. With
the focus that MIST has brought to metabolites, there have been ac-
companying efforts to produce metabolites of interest on such a scale
that they can be readily isolated, quantitated, and profiled. Standard
organic synthesis procedures are frequently unable to produce the
stereo- and regio-specific metabolites of drugs. Biocatalysts offer several
advantages over chemical syntheses because they can often provide
better chemo-, regio-, and stereo-selectivity under milder conditions. As
cytochrome P450 (P450 or CYP) enzymes have poor catalytic efficiency
relative to other enzyme systems, numerous efforts have been under-
taken to engineer the catalytic improvements of P450 s. At present,
most engineered P450 s could not achieve the high catalytic rates
generally observed in bacterial P450 s, coupled with the broad substrate
acceptance of human P450 s. However, researchers have suggested that
the evolution of P450 s can be applied to produce drug metabolites for
drug discovery and development [13].
2.1. Materials
Tenatoprazole (racemic mixture) was purchased from Abcam
Biochemicals (Cambridge, UK). Nicotinamide adenine dinucleotide
+
+
phosphate (NADP ), reduced NADP (NADPH), omeprazole (racemic
mixture), glucose 6-phosphate, and glucose 6-phosphate dehy-
drogenase were purchased from Sigma-Aldrich (St. Louis, MO).
Tenatoprazole sulfide was obtained from 4Chem Laboratory (Suwon,
Gyeonggi-do, Korea). Other chemicals were of the highest grade com-
mercially available.
2
.2. CYP102A1 mutants for screening highly active hydroxylases toward
tenatoprazole
From our previous work to produce human metabolites of drug
molecules and natural products using CYP102A1 mutants, a large set of
CYP102A1 mutant collections has been made [15,25–31]. On the other
hand, the wild-type (WT) CYP102A1 did not show apparent activities
toward the non-natural substrates. We selected 50 mutants from the
mutant collection to screen highly active mutants toward tenatoprazole
In particular, a large set of CYP102A1 mutants are known to have
the ability to produce human metabolites of a number of marketed
drugs and steroids [14–16]. They can produce human metabolites of a
large set of clinical drugs, such as pantoprazole, lansoprazole, rabe-
prazole, simvastatin, lovastatin, fluvastatin, atorvastatin, diclofenac,
tamoxifen, propafenone, astemizole, ibuprofen, tolbutamide, and me-
fenamic acid and other nonsteroidal anti-inflammatory drugs
(
Supplementary Table S1). These mutants were constructed using site-
directed mutagenesis of the active site residues [19,27,28–31] and
error-prone PCR [15,25,26] of the heme domain (1–430 amino acid
residues) of the chimera M16V2. The triple mutant M10 (R47 L/F87 V/
L188Q) was chosen, as this mutant showed a high activity of omepra-
zole 5’-hydroxylation [19]. Mutants M10 and M16 have mutations in
the substrate channel and active site [27]. Each mutant bears amino
acid substitution(s) relative to WT CYP102A1, as summarized in Sup-
plementary Table S1. The M16V2 chimeric fusion protein was obtained
by the reductase domain swapping of the M16 mutant at key catalytic
residues (R47 L/F81I/F87 V/E143 G/L188Q/E267 V) of the heme do-
main with that of a natural variant (CYP102A1.2) of CYP102A1 from
Bacillus megaterium [32]. A DNA library of the chimeric mutants was
obtained by error-prone PCR of the chimera M16V2 heme domain
[
15,17–21]. They also can oxidize various non-natural substrates, such
as natural products and environmental chemicals [16,22,23]. There-
fore, CYP102A1 can be considered a prototype P450 for the versatile
biocatalysts used in drug discovery and synthesis [15,24]. Recently, the
5
’-hydroxylation activity of CYP102A1 mutants on omeprazole [17–19]
and omeprazole sulfide [25] with high selectivity and productivity has
been reported. These results suggest that engineered CYP102A1 can be
used as a biocatalyst to produce the 5’-OH metabolites of omeprazole
[
17–19] and omeprazole sulfide [25] on a scale used for safety testing.
The aim of this study was to find an efficient enzymatic strategy for
(
1–430 amino acid residues). Chimeric mutants were selected based on
their ability to show 4-nitrophenol hydroxylase activity. The selected
chimeric mutants showed high activity toward several typical human
P450 drug substrates, including chlorzoxazone, simvastatin, lovastatin,
fluvastatin, atorvastatin, diclofenac, tamoxifen, propafenone, and as-
temizole [15].
the synthesis of human major metabolites of tenatoprazole using bac-
terial P450 enzymes. Here, we found that CYP102A1 mutants can
catalyze the regioselective hydroxylation of tenatoprazole and tenato-
prazole sulfide at the C-5’ position with a high regioselectivity. To our
knowledge, this is the first report to produce major human metabolites
of tenatoprazole using bacterial P450 s.
The selected mutants were expressed in the Escherichia coli and
partially purified as lysate [15]. The plasmids of WT CYP102A1
(
pCWBM3) and its mutants were transformed into E. coli strain DH5αF-
2

T.-K. Le, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
IQ (ThermoFisher Scientific, Massachusetts, USA), according to the
manufacturer’s instructions. The first culture was inoculated from a
single colony into 5 ml of Luria-Bertani medium, supplemented with
was incubated at 37 °C for 5 min, the formation rate of products was
determined by using HPLC as described above. The kinetic parameters
(kcat and K ) were calculated using a Michaelis-Menten nonlinear re-
M
1
00 μg/ml ampicillin and grown at 37 °C. This culture was used to in-
gression analysis with GraphPad Prism (GraphPad Software, San Diego,
oculate 250 ml of Terrific Broth medium, supplemented with 100 μg/ml
ampicillin. The cells were grown at 37 °C, with shaking at 250 rpm, to
an OD600 ∼ 0.8, at which time gene expression was induced by the
addition of isopropyl-β-D-thiogalactopyranoside (final concentration,
CA).
To determine the total turnover number (TTN) for the four
CYP102A1mutants, 1 mM of tenatoprazole was used. The reaction was
initiated by the addition of the NADPH-generating system and in-
cubated for 30 min, 1 h, and 2 h at 37 °C. The formation rate of 5’-OH
Tenatoprazole, 5’-OH tenatoprazole sulfide, and an unidentified
monohydroxylated product was determined by using HPLC as described
above.
0
.50 mM) with δ-aminolevulinic acid (final concentration, 1.0 mM).
Following induction, the cultures were allowed to grow for another
3
4
6 h at 30 °C. Cells were harvested using centrifugation (15 min, 5000 g,
°C). The cell pellet was resuspended in TES buffer [100 mM Tris-HCl
(
(
pH 7.6), 500 mM sucrose, 0.5 mM EDTA] and lysed via sonication
Sonicator, Heat Systems – Ultrasonic, Inc.). The lysate was centrifuged
2.4. Liquid chromatography-mass spectrometry analysis
at 100,000 g (90 min, 4 °C) to collect the soluble cytosolic fraction, and
it was used for the activity assay. The soluble fraction (so-called “ly-
sate”) was dialyzed against 50 mM potassium phosphate buffer (pH 7.4)
and stored at ˗80 °C. Enzymes were used within 1 month of purification.
CYP102A1 concentrations were determined from the CO-difference
To identify the major metabolites of tenatoprazole produced by
CYP102A1, a liquid chromatography-mass spectrometry (LC–MS) ana-
lysis was performed, and the LC profile and fragmentation patterns of
the authentic compounds (i.e., tenatoprazole and tenatoprazole sulfide)
were compared on a Shimadzu LCMS-2010 EV system (Shimadzu,
Kyoto, Japan) with LC–MS solution software. The hydroxylation reac-
tion of tenatoprazole by CYP102A1 was performed as described above.
The separation was performed on a Shim-pack VP-ODS column
−1
−1
spectra using ε =91 mM cm . For all of the WT and mutated en-
zymes, a typical culture yielded 200 to 700 nM of P450 protein.
2.3. Hydroxylation of tenatoprazole by CYP102A1
(
2.0 × 250 mm; Shimadzu) with a mobile phase of acetonitrile/water
As tenatoprazole has a similar chemical structure to omeprazole, a
(50:50, v/v) at a flow rate of 0.10 ml/min. The mass spectra were re-
corded using electrospray ionization in the positive mode to identify the
metabolites. The nebulization gas flow was set to 1.5 l/min. The in-
terface, curve desolvation line, and heat block temperatures were
250 °C, 230 °C, and 200 °C, respectively.
high-performance liquid chromatography (HPLC) method was based on
an analytical method for omeprazole and its metabolites, as previously
described [19,25]. Racemic mixture of tenatoprazole was used in this
study because each chiral tenatoprazole is not currently available
commercially. The reaction mixture included 0.20 μM CYP102A1 and
Furthermore, to get more detailed information about the metabolites,
the incubation mixtures were analyzed using a 6530 Quadrupole Time-
of-Flight (QTOF) mass spectrometer (Agilent, Santa Clara, CA, USA)
coupled with a 1290 Infinity ultra-performance liquid chromatography
(UPLC) system (Agilent). Separation was performed using a HypersilGold
C18 column (100 × 2.1 mm, 1.9 μm; Thermo, Milford, MA, USA); the
gradient mobile phase was 0.1% (v/v) formic acid in water (A) and 0.1%
(v/v) formic acid in acetonitrile (B), delivered at a flow rate of 0.4 ml/
min. The initial composition of mobile phase B was 10%; this increased
to 30% over 9 min, decreased to 10% for 3 min over 0.2 min, and finally
re-equilibrated to the initial conditions over 0.8 min. Thus, the total run
time was 10 min. The temperatures of the column and autosampler were
kept at 35 °C and 4 °C, respectively, and the injection volumes were 1 μl
for all samples tested here. The electrospray ionization procedure was
performed in the positive ion mode. The sheath gas flow rate was 8 l/
min. The drying gas flow rate was 12 l/min at 350 °C, and the nebulizer
temperature was 350 °C. The capillary voltage was 4500 V in the positive
mode and that of the fragmentor was 175 V. All data were acquired over
0
.10 mM tenatoprazole in 0.25 ml of potassium phosphate buffer
(
0.10 M, pH 7.4). The reaction was started by the addition of an NADPH
+
regeneration system (10 mM glucose-6-phosphate, 0.50 mM NADP
,
and 1.0 IU yeast glucose-6-phosphate dehydrogenase/ml) at 37 °C.
After the indicated time, it was stopped by adding cold ethyl acetate
(
0.50 ml). Omeprazole was added as an internal standard to this solu-
tion at a final concentration of 20 μM [5]. The reaction mixtures were
then vortexed for 2 min. After centrifuging at 3000 rpm for 10 min, the
upper layer was removed for drying under a stream of nitrogen gas.
After the residues were dissolved in 180 μl of mobile phase, 30 μl
samples were injected onto a Gemini C18 column (4.6 × 150 mm, 5 μm;
Phenomenex, Torrance, CA) with an acetonitrile/5 mM potassium
phosphate buffer (pH 7.3) (25:75, v/v) as the mobile phase. Product
formation was analyzed by HPLC at A302
.
Calibration standards of tenatoprazole and omeprazole were con-
structed from a blank sample (a reaction mixture without substrate and
internal standard) and twelve samples (a reaction mixture with tena-
toprazole and omeprazole) covering 1–500 μM. The peak area ratio of
tenatoprazole to the internal standard (omeprazole) was linear with
respect to the analyte concentration over the range of 1–500 μM. When
two volume ethyl acetate was used to extract tenatoprazole and internal
standard in buffer solution, the extraction efficiencies of tenatoprazole
and internal standard were 70% and 74%, respectively, at the con-
centrations used in the assay. Quantitation of the metabolites was done
by comparing the peak areas of each metabolite to the mean peak areas
of the internal standard (20 μM).
a scan range from 50 m/z to 1000 m/z in the centroid mode, and a re-
+
ference compound (C18
H
18
O
6
N
3
P
3
F
24; [M+H] = 922.0098) was used
to correct all masses. Tandem mass spectrometry (MS/MS) data were
acquired in the profile mode (via autoanalysis) for structural character-
ization; the collision energy was 30 eV.
2.5. Identification of major metabolites of tenatoprazole by NMR
spectroscopy
The time course analysis of tenatoprazole hydroxylation using the
mutant enzymes to measure yield of metabolites and percentage of each
metabolite was determined by using 0.20 μM CYP102A1 with 0.10 mM
tenatoprazole in 0.25 ml of potassium phosphate buffer (0.10 M, pH
To identify two hydroxylated metabolites, they were separated
using HPLC and collected. The reaction mixture contained 0.4 μM of
CYP102A1 M371 and 1 mM of tenatoprazole in 30 ml of potassium
phosphate buffer (0.10 M, pH 7.4). An NADPH regeneration system was
used to start the reaction. The sample was incubated at 37 °C for 2 h.
Product formation was separated by using analytical HPLC as described
above. Isolated yields of 5’-OH tenatoprazole and 5’-OH tenatoprazole
sulfide were 8.2% and 1.3%, respectively. NMR experiments were
performed at 25 °C on a Varian VNMRS 600 MHz NMR spectrometer
7
.4). An NADPH regeneration system was used to start the reaction. The
samples were incubated at 37 °C for 2, 5, 10, 30, 60, and 120 min.
Product formation was analyzed using HPLC, as described above.
To analyze the steady-state kinetics (kcat and K ) of each CYP102A1
M
mutant, the reaction mixture contained 0.20 μM mutant enzyme, an
NADPH-generating system, and tenatoprazole (10–500 μM). After it
equipped with a carbon-enhanced cryogenic probe. DMSO-d was used
6
3

T.-K. Le, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
Fig. 2. (a) The metabolic pathway of tenatoprazole via the
CYP102A1 mutant to produce human metabolites. It starts
from tenatoprazole (1). There are two metabolites of te-
natoprazole: 5’-OH tenatoprazole (2), produced by the
CYP102A1 mutant, and tenatoprazole sulfide (4), which is
formed non-enzymatically. The last product in the pathway
is 5’-OH tenatoprazole sulfide (3), which comes from te-
natoprazole sulfide via CYP102A1 and/or 5’-OH tenato-
prazole via non-enzymatic conversion. The structures were
created using ACD/Chemsketch (https://www.acdlabs.
com). (b) HPLC chromatograms of the tenatoprazole me-
tabolites produced by mutant M371 with (and without)
NADPH. The reaction mixture containing P450 (0.20 μM)
and tenatoprazole (0.10 mM) was incubated at 37 °C for
1
0 min. (1) Tenatoprazole: t
toprazole: t =3.05 min; (3) 5’-OH tenatoprazole sulfide: t
4.90 min; (4) Tenatoprazole sulfide: t =43.9 min; (*)
R
=6.82 min; (2) 5’-OH tena-
R
R
=
R
R
internal standard omeprazole (20 μM): t =14.5 min. The
inset of panel (b) shows the peak of tenatoprazole sulfide
using magnification.
as a solvent, and chemical shifts for proton NMR spectra were measured
in parts per million (ppm) relative to tetramethylsilane. All of the NMR
experiments were carried out with standard pulse sequences in VNMRJ
3. Results and discussion
3.1. Hydroxylation of tenatoprazole by CYP102A1
(
v. 3.2) library and processed with the same software.
We used the WT enzyme and 50 mutants with substituted amino
acid residue(s) in the substrate channel and active site to determine
whether CYP102A1 can hydroxylate tenatoprazole. Tenatoprazole hy-
droxylation activity was examined at a fixed substrate concentration of
100 μM for 30-min incubation. A major hydroxylation product is 5’-OH
tenatoprazole, and a minor product is 5’-OH tenatoprazole sulfide
(Fig. 2). Tenatoprazole sulfide is another major product that is formed
non-enzymatically, even in the absence of P450 and NADPH (Fig. 2b).
Among all enzymes tested here, only 26 mutants (changed amino acid
sequences are shown at Supplementary Table S1) showed apparent
activities of hydroxylation (Fig. 3). In the case of the WT, any mea-
2
.6. NADPH oxidation
The reaction was performed in a spectrophotometric cuvette
maintained at 37 °C. The reaction mixture contained 0.10 μM of
CYP102A1 and 0.50 mM of tenatoprazole in 1 ml of potassium phos-
phate buffer (0.10 M, pH 7.4). The reactions were initiated by adding
1
0 μl of 10 mM NADPH (final concentration, 100 μM), and the decrease
in A340 was monitored for 1 min. The rates of NADPH oxidation were
calculated using ε340 = 6.22 M cm for NADPH.
−
1
−1
−1
2
.7. Spectral binding titration
surable activity was not observed (lower than 0.01 min ). Most of the
active mutants showed two major hydroxylated products and tenato-
prazole sulfide. On the other hand, mutant M10, chimera M16V2
(which the reductase domain of mutant M16 was replaced with the
reductase domain of a natural variant V2 of CYP102A1), and two chi-
meric mutants of M16V2 (M413 and M416) [15] only showed one
major hydroxylation product (5’-OH tenatoprazole). Although the ac-
All spectrophotometric measurements were recorded with
a
Shimadzu 1601PC Spectrophotometer at 23 °C. The binding affinity of
ligands to the CYP102A1 enzymes was determined by titrating 1.5 μM
of the enzyme with the ligand in a total volume of 1.0 ml of 100 mM
potassium phosphate buffer (pH 7.4). The final CH CN concentration
3
−1
was < 2% (v/v). The reference cuvette, containing an equal con-
centration of the enzyme in the buffer, was titrated with an equal vo-
lume of the vehicle solvent. UV–vis spectra (350–500 nm) were re-
corded after each addition, and the absorbance differences were plotted
against the added ligand concentrations. Spectral dissociation constants
tivity of most mutants is less than 1 min , two chimeric mutants
(M371 and M387) showed the highest turnover numbers of 5’-OH te-
−1
natoprazole (3.43 and 1.72 min , respectively) and 5’-OH tenatopra-
zole sulfide (0.33 and 0.29, respectively) among all the tested mutants.
The identities of the metabolites and substrate were confirmed using
LC–MS (Supplementary Fig. S1-S2) and NMR analyses (Supplementary
Fig. S3-S7).
(
K indicates a spectrally estimated dissociation constant) were esti-
D
mated as previously described [27,33,34] using GraphPad Prism soft-
ware (GraphPad software, San Diego, CA). Because of the high affinities
Spectral assignments of tenatoprazole and its metabolites, 5’-OH
tenatoprazole (M1) and 5’-OH tenatoprazole sulfide (M2), were carried
of tenatoprazole (K within 5-fold of the P450 concentration), a non-
D
1
linear regression analysis using a quadratic equation was applied to
out with H NMR, COSY, and 1-dimensional selective NOE experiments
determine the K
D
for tenatoprazole. With tenatoprazole sulfide, the best
(Supplementary Fig. S3-S7). Chemical shift assignments for tenatopra-
fit was obtained using the Hill equation, as steady-state binding titra-
tion data were sigmoidal [33,34].
zole (M0) in DMSO-d was reported [35]. However, we found some
6
discrepancies in the assignments for the H-6 and H-7 protons of
4

T.-K. Le, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
Fig. 4. Time course profile of tenatoprazole and its metabolites using mutants
M371 (a) and M387 (b). Yields of each compound at the indicated reaction time
were determined by using 0.20 μM CYP102A1 with 0.10 mM tenatoprazole in
0.25 ml of potassium phosphate buffer (0.10 M, pH 7.4). The samples were
incubated at 37 °C for 2, 5, 10, 30, 60, and 120 min. Control experiments
(dotted lines) in the absence of NADPH were done at the same experimental
conditions. The values are presented as the means ± SD of triplicate mea-
surements.
Fig. 3. Formation rate of the tenatoprazole metabolites catalyzed by CYP102A1
mutants. The formation rates of the products were determined using HPLC, as
described in Materials and Methods. The values are presented as the
means ± SD of triplicate measurements. (a) 5’-OH tenatoprazole; (b) 5’-OH
tenatoprazole sulfide; (c) uncharacterized monohydroxylated metabolite; (d)
tenatoprazole sulfide.
1
00 μM. For mutant #371 (Fig. 4a), the substrate tenatoprazole gra-
tenatoprazole after running a series of 1-dimensional selective NOE
dually decreased during the reaction time, and 7.5% of tenatoprazole
remained after a 2 h reaction. At the same time, the major product, 5’-
OH tenatoprazole, reached the maximal yield (18%) after a 60 min
reaction. Tenatoprazole sulfide and 5’-OH tenatoprazole sulfide were
raised slightly (1.4% and 2.0% yield, respectively, after a 60 min in-
cubation). However, the time course analysis of M387 (Fig. 4b) showed
a distinct result when compared to that of M371. In the case of M387,
tenatoprazole also gradually decreased to 14% yield after a 2 h in-
cubation, while the rate of tenatoprazole sulfide formation was much
faster than that of variant M371. Consequently, the highest percentage
yield of 5’-OH tenatoprazole by M387 for a 30 min reaction was 6.9%,
which is only one-third of the tenatoprazole sulfide formation by M371.
Although tenatoprazole sulfide gradually increased with incubation
time, reaching to 17% at 2 h of incubation, 5’-OH tenatoprazole sulfide
only increased to 1.1%. The ratio of products as percentage after the 2 h
reaction at neutral pH was shown at Supplementary Table S3.
1
experiments (Supplementary Fig. S6). Correct H NMR chemical shifts
of TEN (M0) and its metabolites (M1 and M2) are listed in Supple-
mentary Table S2. Important NOE results for the identification of te-
natoprazole and its metabolites are shown in Supplementary Fig. S6.
Another metabolite (at t = ∼10 min) was observed at the HPLC
R
trace of all tested samples. As this metabolite is shown only in the pre-
sence of NADPH, it is also a metabolite of the CYP102A1-catalyzed re-
action. UPLC-ESI-QTOFMS analysis has been done to obtain more in-
formation about metabolites, including another major unidentified
metabolite (so-called “metabolite 5,” seen in Supplementary Fig. S2). The
MS spectra analyses show that the metabolites of tenatoprazole include
5
’-OH tenatoprazole, 5’-OH tenatoprazole sulfide, tenatoprazole sulfide,
two monohydroxylated products, and tenatoprazole sulfone. The tena-
toprazole sulfone was also detected, although the sulfone was not de-
tected in the HPLC trace. We suggest that the unidentified metabolite
(
metabolite 5) is a monohydroxylated product at the imidazopyridine
We examined whether tenatoprazole sulfide formation, a reduction
reaction of sulfoxide to sulfide, is catalyzed by a P450 system (P450
heme or reductase domain) or transformed non-enzymatically. When
the concentrations of tenatoprazole and its sulfide were measured with
the reaction mixtures of both mutants without NADPH, the tenatopra-
zole concentration rapidly decreased to 35% and 42% in 10 min for the
mutants M371 and M387, respectively (Fig. 4). After that, its con-
centrations gradually decreased with incubation time. After 2 h of in-
cubation, only 26% and 23% of tenatoprazole remained for the mutants
M371 and M387, respectively. However, at the same time, the tenato-
prazole sulfide concentration increased to a small extent during the
incubation time. After 2 h of incubation, only 7.5% and 6.7% of tena-
toprazole sulfide were formed for the mutants M371 and M387, re-
spectively (Fig. 4 and Supplementary Table S3).
ring of tenatoprazole (Supplementary Fig. S2 b, c, and h). Although some
−1
mutants show high turnover numbers (up to 3.9 min ), which were
calculated based on the area of internal standard, for the formation of
this monohydroxylated product, the product showed a very low detec-
tion level in the mass spectrometer. The extent of this metabolite’s for-
mation was variable among the mutant CYP102A1 lines, similar to the
other metabolites described herein (Fig. 3c). However, the chemical
structure of this metabolite was not identified in this study.
3
.2. Time course analysis of hydroxylated metabolites of tenatoprazole by
CYP102A1 mutants
Mutants M371 and M387, which exhibited high product formation
rates, were selected for time course study and additional kinetic ana-
lyses. Time course measurement with tenatoprazole was performed at
To investigate whether the non-enzymatic reduction of tenatopra-
zole to the sulfide requires the bacterial lysate, we only measured the
5

T.-K. Le, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
Table 1
The steady-state kinetics of hydroxylated product formation from tenatoprazole using CYP102A1 mutants.
5’-OH tenatoprazolea
5’-OH tenatoprazole sulfide
b
Monohydroxylated metabolitec
CYP102A1
k
cat (min−1)
K
M
(μM)
kcat/K
M
k
cat (min−1
)
K
M
(μM)
kcat/K
M
k
cat (min−1
)
K
M
(μM)
kcat/K
M
M371
M387
16 ±
1
210 ± 20
98 ± 28
0.076
0.054
1.3 ± 0.1
230 ± 10
180 ± 50
0.0057
0.0047
17 ±
1
550 ± 20
70 ± 20
0.031
0.046
5.3 ± 0.6
0.84 ± 0.10
b
3.2 ± 0.3
The steady-state kinetics (kcat and K
M
) of a5’-OH tenatoprazole and 5’-OH tenatoprazole sulfide formation using CYP102A1 mutants were determined in a final
volume of 0.25 ml of potassium phosphate buffer (0.10 M, pH 7.4). The reaction mixture contained 0.20 μM enzyme, an NADPH regeneration system, and tena-
toprazole (10–500 μM). The samples were incubated for 5 min at 37 °C. Data represent mean values ± SD from triplicate measurements.
formation of tenatoprazole sulfide from tenatoprazole (100 μM) in a
buffer solution (100 mM potassium phosphate, pH 7.4) (Supplementary
Fig. S8). While the tenatoprazole gradually decreased during the in-
cubation time, the tenatoprazole sulfide concentration increased to a
small extent. After 10 and 30 min of incubation, 8% and 20% of tena-
toprazole were broken down, respectively, although only 1% and 2% of
tenatoprazole sulfide were formed. After 2 h of incubation, 45% of te-
natoprazole remained and 6% of tenatoprazole sulfide was formed.
After 8 h and 12 h, 96% and 99% of tenatoprazole were decomposed,
respectively. After more than 8 h of incubation, most of tenatoprazole
was decomposed, and only 20% of tenatoprazole sulfide remained. This
result indicates that tenatoprazole is intrinsically unstable, even in a
neutral buffer solution.
3.3. Kinetic parameters of 5’-OH tenatoprazole and 5’-OH tenatoprazole
sulfide formation by CYP102A1 mutants
Table 1 and Supplementary Fig. S10 show the steady-state kinetics
of hydroxylated product formation from tenatoprazole by mutants
M371 and M387. In the case of 5’-OH tenatoprazole formation, mutant
M371 had higher kcat value than that of mutant M387 (∼ 3-fold). By
contrast, the value of K by mutant M387 (98 μM) was 2.1-fold lower
M
than that of mutant M371 (210 μM). The kcat of mutant M371 for 5’-OH
−1
tenatoprazole sulfide formation was 1.3 min , which is 1.5-fold of that
by mutant M387. Data show that 5’-OH tenatoprazole formation ex-
hibited significantly higher kcat value of each enzyme than that of 5’-OH
tenatoprazole sulfide formation by 6∼12-fold. In addition, K values of
M
After 2 h of incubation of tenatoprazole with P450 containing lysate
and NADPH, about 50% of tenatoprazole was transformed to other
forms of metabolites; 5’-OH tenatoprazole, 5’-OH tenatoprazole sulfide,
M371 for 5’-OH tenatoprazole and 5’-OH tenatoprazole sulfide forma-
tion were higher than those of M387. Catalytic efficiencies of 5’-OH
tenatoprazole formation by two mutants were much higher than those
of 5’-OH tenatoprazole sulfide formation by 11∼13-fold (Table 1).
However, a direct comparison of the kinetic parameters to tenatopra-
zole and its sulfide form is not possible because the exact concentration
of tenatoprazole sulfide cannot be determined in this experiment. This
result indicates that 5’-OH tenatoprazole formation is the major reac-
tion when tenatoprazole is used as a substrate.
a
monohydroxylated tenatoprazole, and tenatoprazole sulfide
(
Supplementary Table S3). The other 50% of tenatoprazole seemed to
convert to other metabolites. However, incubation of tenatoprazole
with P450 containing lysate without NADPH accelerates the decom-
position of tenatoprazole. After 2 h of incubation, ∼30-34% tenato-
prazole remained, and only ∼7% tenatoprazole sulfide was formed.
The formation of the sulfide occurred with or without NADPH;
however, the rate of sulfide formation for mutant M387 was greater
with NADPH (Fig. 4b). Conversely, for mutant M371, the rate of sulfide
formation was much lower with NADPH than without NADPH (Fig. 4a).
The sulfide formation in mutant M371 seems to be actively suppressed.
We found that the formation of tenatoprazole sulfide is mainly non-
enzymatic and does not need NADPH (Fig. 4). Therefore, some portion
of tenatoprazole will be converted to a sulfide form at a rapid pace
when tenatoprazole is administered. This result suggests a possibility
that tenatoprazole sulfide can be used as a substrate for P450-catalyzed
reaction. There is another possibility: 5’-OH tenatoprazole sulfide is
non-enzymatically formed from 5’-OH tenatoprazole. When considered
together, the non-enzymatic decomposition of tenatoprazole to tena-
toprazole sulfide at a neutral pH should be considered for the study
metabolism of tenatoprazole during the drug development process.
On the other hand, omeprazole seems to be relatively stable at a
neutral pH because omeprazole was reported to be stable at pH 7.5 for
When the kinetic parameters of an unidentified monohydroxylated
product formation by CYP102A1 mutants were determined, the kcat
values of two mutants were comparable to those of 5’-OH tenatoprazole
formation. The K
M
value of M371 for unidentified monohydroxylated
product formation was 2.6-fold higher than that of 5’-OH tenatoprazole
formation. However, direct comparison of the kinetic parameters be-
tween 5’-OH tenatoprazole formation and unidentified mono-
hydroxylated product formation is not possible because the exact con-
centration of the unidentified monohydroxylated product cannot be
determined in this experiment. The results indicate that the uni-
dentified monohydroxylated product is also a major metabolite, which
is produced from tenatoprazole catalyzed by P450.
When the TTNs (nmol product/nmol P450) of M371 for the for-
mation of 5’-OH tenatoprazole and the unidentified monohydroxylated
product were determined with 1 mM tenatoprazole during the incuba-
tion of 30 min, 1 h, and 2 h, the overall range was 200–380
(Supplementary Fig. S11). While the formation of 5’-OH tenatoprazole
decreased after 30 min of incubation, that of the unidentified metabo-
lite formation increased with incubation time. This indicates that 5’-OH
tenatoprazole can be converted to other metabolites. However, the
TTNs of 5’-OH tenatoprazole sulfide formation was only 20–24. The
TTNs of M387 were much lower than those of M371 for the formation
of three metabolites. The TTNs for the 5’-OH tenatoprazole formation
(120∼382) by CYP102A1 mutants are much lower than those for the
5’-OH omeprazole (950∼1200) [19].
2
4 h [36]. However, omeprazole is known to be unstable under acidic
conditions, in which it easily degraded [8,37,38]. Omeprazole in aqu-
eous solutions between pH 2.2 and pH 4.7 degraded completely in less
than 4 h [38]. As a control experiment, the stability of omeprazole at
pH 7.4 and pH 5.0 was compared (Supplementary Fig. S9). As ome-
prazole is stable at pH 7.4, it remains 99% and 95% after incubation for
2
h and 5 h, respectively. On the other hand, 93% and 99% of ome-
prazole degraded after 2 h and 5 h of incubation at pH 5.0, respectively.
During the degradation of omeprazole at pH 5.0, 32% and 38% of
omeprazole sulfide was formed for 2 h and 5 h, respectively. Several
other degradation product peaks in addition to the peak of omeprazole
sulfide were also observed during incubation under this acidic condi-
tion. This result is consistent with previous reports regarding the sta-
bility of omeprazole under acidic conditions.
When the rates of product formation and NADPH oxidation were
compared, M387 (1.8%) showed lower coupling efficiency than M371
(3.3%) toward 0.5 mM tenatoprazole (Table 2). The coupling efficiency
of the tenatoprazole oxidation is much lower than other non-natural
substrates, such as omeprazole sulfide (16–82%) [25], testosterone
(14–84%) [39], and norethisterone (14–67%) [39].
6

T.-K. Le, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
Table 2
Catalytic properties of tenatoprazole oxidation using CYP102A1 mutants.
CYP102A1
Producta
NADPH oxidation (min−1)b
Product formation (min−1)c
Coupling efficiency (%)d
TTNe (reaction time)
#
371
389 ± 21
3.3
5
5
’-OH tenatoprazole
8.02 ± 0.24
0.64 ± 0.03
4.39 ± 0.05
2.06
0.16
1.13
1.8
383 ± 4 (0.5 h)
24 ± 1 (0.5 h)
313 ± 12 (2 h)
’-OH tenatoprazole sulfide
Monohydroxylated metabolite
#387
445 ± 24
5
5
’-OH tenatoprazole
4.37 ± 0.35
0.54 ± 0.02
2.97 ± 0.10
0.98
0.12
0.67
120 ± 3 (0.5 h)
8.4 ± 0.2 (0.5 h)
77 ± 8 (2 h)
’-OH tenatoprazole sulfide
Monohydroxylated metabolite
a
As shown, 5’-OH tenatoprazole, 5’-OH tenatoprazole sulfide, and monohydroxylated metabolite are considered to be the major metabolites when using
CYP102A1 mutants.
b
c
d
e
The NADPH consumption rates were measured over 1 min at 340 nm as nmol NADPH/min/nmol P450.
The product formation rates were determined as nmol-indicated product/min/nmol P450 using tenatoprazole (0.2 mM) in the Materials and Methods section.
The corresponding coupling efficiency is defined as the rate of product formation divided by the rate of NADPH consumption.
The TTNs for the hydroxylation of omeprazole sulfide were determined as described in the Materials and Methods section. Data represent the mean values ± SD
from triplicate measurements.
3
.4. Binding titration of tenatoprazole and tenatoprazole sulfide toward
suggesting a high degree of binding cooperativity. When the values of
CYP102A1 mutants
K
d
d
and K
M
of tenatoprazole to the mutants were compared, the values of
K
were much lower than those of K
M
. Even though the tenatoprazole
The mutants M371 and M387 contain a mixture of low- and high-
spin heme iron (Supplementary Fig. S12). The addition of tenatoprazole
and tenatoprazole sulfide to a solution with the mutants M371 and
M387 produced difference spectra of reverse Type I and Type I, re-
spectively (Fig. 5 and Supplementary Fig. S12). The binding affinity of
the mutants toward the tenatoprazole and tenatoprazole sulfide sub-
strates was determined from the titration curves. The mutants have a
can tightly bind to the active site at a very low concentration of tena-
toprazole, the oxidation reactions to make products do not seem to
proceed easily. On the other hand, the addition of tenatoprazole or
tenatoprazole sulfide to a WT solution did not cause any apparent
spectral changes.
When considered together, the formation of 5’-OH tenatoprazole
sulfide when tenatoprazole was used as a substrate suggests the possi-
bility of two metabolic pathways of tenatoprazole. After 5’-OH tena-
toprazole is produced by CYP102A1, it can be non-enzymatically
transformed to 5’-OH tenatoprazole sulfide. On the other hand, tena-
toprazole sulfide, a major metabolite of tenatoprazole, can be used as a
higher affinity for tenatoprazole (K = 0.89∼1.0 μM) than tenatopra-
d
zole sulfide (K = 105∼124 μM). The titration plots obtained for te-
d
natoprazole sulfide binding to the mutants displayed apparent sigmoi-
dicity. A fit to the Hill equation resulted in coefficients (n) of 1.7–1.9,
Fig. 5. Spectral binding titration of CYP102A1 with tenatoprazole and tenatoprazole sulfide. CYP102A1 (1.5 μM) was titrated as a function of ligand concentration in
00 mM potassium phosphate (pH 7.4), as described in Materials and Methods: mutants #371 (a and c); #387 (b and d) with tenatoprazole (a and b) and
tenatoprazole (c and d). The inset of each panel shows a plot of induced Soret absorbance change (ΔA390-420) versus the relevant concentration of ligands for mutants
M371 and M387. Dissociation constants (K ) for the ligand binding and maximal changes of absorption (Bmax) were shown. Spectral changes of the heme iron from
the unbound to bound substrate were marked with arrows.
1
d
7

T.-K. Le, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
substrate for a hydroxylation pathway after non-enzymatic transfor-
mation to tenatoprazole sulfide from tenatoprazole (Fig. 2a).
methods can be used to improve upon this low level of activity and
regio-selectivity to produce the metabolites on a scale to meet the MIST
issue [13].
When the hydroxylation activities of mutant M10 toward C-5’ of
tenatoprazole (this work) and omeprazole [19] were compared, this
In recent years, P450-catalyzed reactions have been of special in-
terest for selective C–H oxygenation to produce fine chemicals and
pharmaceuticals [42–44]. For industrial fine chemical production,
biotransformations have been considered to require a minimum space-
−1
mutant showed much higher activity of omeprazole (52 min ) than
−
1
that of tenatoprazole (0.44 min ). Mutant M10 is a well-known,
highly active mutant toward several human substrates and has muta-
tions in the substrate channel and active site (R47 L/F87 V/L188Q)
−
1
−1
time yield (0.1 g l
h
) and a minimum final product concentration
−1
−1
[
40]. However, the highest active mutant, M371 (kcat, 16 min ), to-
(1 g l ). For industrial pharmaceutical production, an efficient time-
to-market strategy is more crucial than the production costs, with es-
timated minimum process requirements for volumetric productivity
ward tenatoprazole has additional mutations in the substrate channel
and active site (D23 G, F81I, F107 L, E143 G, and E267 V) and outside
the active site and substrate channel (D136 G). Although M371 shows
the highest activities for producing three major metabolites among
tested mutants, M389, M413, and M416 show very low activities for
producing the metabolites. M16, H1, M179, and M221 show a re-
gioselective preference for the unidentified monohydroxylated meta-
bolite, producing 5’-OH tenatoprazole (Fig. 3). Among the additional
mutation sites for M16, M371, and M387 mutants, D23 is located at the
entrance of the substrate access channel. F107 is located at the active
site under the heme. However, F11, Q110, R190, and D136 are located
outside the substrate access channel and active site. The mutations at
the M371 and M387 appear to have different effects on the con-
formation of the active site upon binding to the substrates (Supple-
mentary Fig. S13). It is well-known that the catalytic rate in enzymes
−1
−1
−1
(0.001 g l
h
) and product concentration (0.1 g l ) [42,45]. Effi-
cient and time-saving production seems to be important in fulfilling the
minimal requirements for industrial pharmaceutical production. At
present, several approaches using engineered CYP102A1 as biocatalysts
seem to achieve the industrially relevant product concentrations. The
Urlacher’s group reported that the productivity of (+)‐nootkatone was
−
1
−1 −1
up to 360 mg l
and had a space‐time yield of 18 mg l
h
for
synthesis of the (+)‐nootkatone from (+)‐valencene using a CYP102A1
variant and an alcohol dehydrogenase (ADH) in one pot [46]. A com-
bination of engineered CYP102A1, ADH, and engineered cyclohex-
−
1
anone monooxygenase could produce final concentrations of ∼3 g l
enantholactone (2‐oxocanone) from cycloheptane [47]. Another report
–
1
described the L-Tyrosine derivatives up to 5.2 g l were being obtained
using an engineering CYP102A1 variant and tyrosine phenol lyase using
(
e.g., dihydrofolate reductase) is significantly affected by mutations far
from the site of chemical activity in the enzyme [41]. Note that mu-
tations at a small number of residues can significantly impact the
structural organization of CYP102A1 and can induce major alterations
in substrate selectivity [18]. When considered together, it can be sug-
gested that combinatorial approaches, including random mutagenesis,
rational design, and target-directed mutagenesis based on the crystal
structure of CYP102A1 with substrates, would be necessary to make
desired enzymes with high activity and regioselectivity.
substituted benzenes, pyruvate, and NH as starting materials [48].
3
4
. Conclusion
In this study, we found that the major hydroxylation product of
tenatoprazole by CYP102A1 is 5’-OH tenatoprazole, a major human
metabolite produced by human CYP2C19 and CYP3A4. Tenatoprazole
sulfide, another major metabolite, is formed via a non-enzymatic con-
version without P450 system. Because tenatoprazole sulfide can be used
as a substrate for a regioselective hydroxylation reaction at C-5’ by
CYP102A1, 5’-OH tenatoprazole sulfide can be produced as a minor
metabolite via CYP102A1 reaction. On the other hand, 5’-OH tenato-
prazole sulfide can also be formed non-enzymatically from 5’-OH te-
natoprazole. Here, we report the enzymatic one-step production of 5’-
OH tenatoprazole from tenatoprazole, which was developed using the
CYP102A1 mutant as a biocatalyst. The tenatoprazole metabolites can
be used for other studies during the drug-development process, such as
drug efficacy, safety, metabolism, and synthesis of derivatives. An en-
zymatic regioselective CeH hydroxylation strategy should be useful for
the production of hydroxylated metabolites of other PPI drugs with
similar chemical structures.
When considered together, CYP102A1 mutants show different pre-
ferences regarding the substrates for the additional hydroxylation at the
imidazopyridine ring in addition to the hydroxylation at the C-5’ po-
sition of the pyridine ring, although tenatoprazole has a similar che-
mical structure to that of omeprazole. As tenatoprazole has a very si-
milar chemical structure to omeprazole, differences in electronic
distribution between the imidazopyridine ring of tenatoprazole and the
benzimidazole moiety of omeprazole might cause the hydroxylation of
tenatoprazole at the imidazopyridine ring. Our results in this study
suggest the possibility of the different metabolic pathways between
tenatoprazole and omeprazole.
Because the chemical synthesis of 5’-OH tenatoprazole is not cur-
rently possible, it should be obtained with human P450 enzymes or
liver microsomes. In this study, engineered bacterial CYP102A1 en-
zymes showed the enhanced catalytic activity of tenatoprazole 5’-hy-
droxylation, and they increased the product yield of 5’-OH tenatopra-
zole. It is now possible to study the effects of 5’-OH tenatoprazole and
Contributors
CHY conceived the project, and procured grants, supervised the
project, and reviewed the data. TKL, GSC, HHJ, THHN, TTMD, YJL,
KDP, YS, and DHK, along with CHY, designed the experiments, and
executed all the experiments and analyzed the data. TKL and CHY
wrote the manuscript.
5
’-OH tenatoprazole sulfide on gastric acid hypersecretion disorders in
in vitro, in vivo, and animal model systems because scalable production
of the metabolites is possible by using bacterial P450. Specifically, the
effects of the 5’-OH metabolites from racemic, S-, and R-tenatoprazole
on several diseases caused by gastric acid hypersecretion are of parti-
cular interest. The effects of these metabolites on gastric acid hy-
persecretion disorders may suggest that the metabolites could be used
as “drug leads” to avoid interindividual variations in drug-metabolizing
enzymes and drug–drug interactions. Furthermore, the metabolites can
be subjected to further structural modifications to obtain improved
clinical candidates during the lead optimization phase of the drug dis-
covery process.
Declaration of Competing Interest
The authors declare that they have no conflict of interest. All au-
thors have approved the manuscript.
Acknowledgments
Although mutant M371 show a high kcat value (16 min−1) and a
high yield (41%) among tested mutants, this P450 enzyme shows re-
latively low levels of activity when compared to a set of industrial
biocatalysts [42]. Directed evolution and other protein engineering
This research was supported by the National Research Foundation
of Korea [Grant NRF-2016R1A2B4006978 and 2018R1A4A1023882],
Republic of Korea.
8

T.-K. Le, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
Appendix A. Supplementary data
[21] L. Capoferri, R. Leth, E. ter Haar, A.K. Mohanty, P.D. Grootenhuis, E. Vottero,
J.N. Commandeur, N.P. Vermeulen, F.S. Jørgensen, L. Olsen, D.P. Geerke, Insights
into regioselective metabolism of mefenamic acid by cytochrome P450 BM3 mu-
tants through crystallography, docking, molecular dynamics, and free energy cal-
culations, Proteins 84 (2016) 383–396, https://doi.org/10.1002/prot.24985.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.procbio.2019.09.014.
[
22] H.M. Girvan, T.N. Waltham, R. Neeli, H.F. Collins, K.J. McLean, N.S. Scrutton,
D. Leys, A.W. Munro, Flavocytochrome P450 BM3 and the origin of CYP102 fusion
species, Biochem. Soc. Trans. 34 (2006) 1173–1177, https://doi.org/10.1042/
BST0341173.
References
[
[
[
1] H. Li, L. Meng, F. Liu, J.F. Wei, Y.Q. Wang, H+/K+-ATPase inhibitors: a patent
review, Expert Opin. Ther. Pat. 23 (2013) 99–111, https://doi.org/10.1517/
[23] L.O. Narhi, A.J. Fulco, Characterization of a catalytically self-sufficient 119,000-
dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus
megaterium, J. Biol. Chem. 261 (1986) 7160–7169.
1
3543776.2013.741121.
2] R.H. Hunt, C. Scarpignato, Potent acid suppression with PPIs and P-CABs: what’s
new? Curr. Treat. Options Gastroenterol. 16 (2018) 570–590, https://doi.org/10.
[24] V.B. Urlacher, M. Girhard, Cytochrome P450 monooxygenases: an update on per-
spectives for synthetic application, Trends Biotechnol. 30 (2012) 26–36, https://
doi.org/10.1016/j.tibtech.2011.06.012.
1
007/s11938-018-0206-y.
[
25] H.H. Jang, S.H. Ryu, T.K. Le, T.T. Doan, H.T. Nguyen, K.D. Park, D.E. Yim,
D.H. Kim, C.K. Kang, T. Ahn, H.S. Kang, C.H. Yun, Regioselective C-H hydroxylation
of omeprazole sulfide by Bacillus megaterium CYP102A1 to produce a human
metabolite, Biotechnol. Lett. 39 (2017) 105–112, https://doi.org/10.1007/s10529-
016-2211-3.
3] R.H. Hunt, D. Armstrong, M. Yaghoobi, C. James, The pharmacodynamics and
pharmacokinetics of S-tenatoprazole-Na 30 mg, 60 mg and 90 mg vs. esomeprazole
4
0 mg in healthy male subjects, Aliment. Pharmacol. Ther. 31 (2010) 648–657,
https://doi.org/10.1111/j.1365-2036.2009.04219.x.
[
4] R.H. Hunt, D. Armstrong, C. James, S.K. Chowdhury, Y. Yuan, P. Fiorentini,
A. Taccoen, P. Cohen, Effect on intragastric pH of a PPI with a prolonged plasma
half-life: comparison between tenatoprazole and esomeprazole on the duration of
acid suppression in healthy male volunteers, Am. J. Gastroenterol. 100 (2005)
[
26] T.K. Le, H.H. Jang, H.T. Nguyen, T.T. Doan, G.Y. Lee, K.D. Park, T. Ahn, Y.H. Joung,
H.S. Kang, C.H. Yun, Highly regioselective hydroxylation of polydatin, a resveratrol
glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450
BM3, Enzyme Microb. Technol. 97 (2017) 34–42, https://doi.org/10.1016/j.
enzmictec.2016.11.003.
1
949–1956, https://doi.org/10.1111/j.1572-0241.2005.41956.x.
[
5] F. Domagala, H. Ficheux, G. Houin, J. Barré, Pharmacokinetics of tenatoprazole, a
newly synthesized proton pump inhibitor, in healthy male Caucasian volunteers,
Arzneimittelforschung 56 (2006) 33–39, https://doi.org/10.1055/s-0031-1296698.
6] S. Yamashita, K. Ebina, Enantiomer of tenatoprazole and the use of thereof in
therapy, US patent 7,507746B2 (2009).
[27] D.H. Kim, K.H. Kim, D.H. Kim, K.H. Liu, H.C. Jung, J.G. Pan, C.H. Yun, Generation
of human metabolites of 7-ethoxycoumarin by bacterial cytochrome P450 BM3,
Drug Metab. Dispos. 36 (2008) 2166–2170, https://doi.org/10.1124/dmd.108.
021220.
[
[
[
28] D.H. Kim, T. Ahn, H.C. Jung, J.G. Pan, C.H. Yun, Generation of the human meta-
bolite piceatannol from the anticancer-preventive agent resveratrol by bacterial
cytochrome P450 BM3, Drug Metab. Dispos. 37 (2009) 932–936, https://doi.org/
7] X.Q. Li, L. Weidolf, R. Simonsson, T.B. Andersson, Enantiomer/enantiomer inter-
actions between the S- and R-isomers of omeprazole in human cytochrome P450
enzymes: major role of CYP2C19 and CYP3A4, J. Pharmacol. Exp. Ther. 315 (2005)
1
0.1124/dmd.108.026484.
7
77–787, https://doi.org/10.1124/jpet.105.090928.
[
[
8] N.L. Rezk, K.C. Brown, A.D. Kashub, A simple and sensitive bioanalytical assay for
simultaneous determination of omeprazole and its three major metabolites in
human blood plasma using RP-HPLC after a simple liquid-liquid extraction proce-
dure, J. Chromatogr. B. 844 (2006) 314–321, https://doi.org/10.1016/j.jchromb.
[29] K.H. Kim, J.Y. Kang, D.H. Kim, S.H. Park, S.H. Park, D. Kim, K.D. Park, Y.J. Lee,
H.C. Jung, J.G. Pan, T. Ahn, C.H. Yun, Generation of human chiral metabolites of
simvastatin and lovastatin by bacterial CYP102A1 mutants, Drug Metab. Dispos. 39
(2011) 140–150, https://doi.org/10.1124/dmd.110.036392.
[
30] D.H. Kim, K.H. Kim, D. Kim, H.C. Jung, J.G. Pan, Y.T. Chi, T. Ahn, C.H. Yun,
Oxidation of human cytochrome P450 1A2 substrates by Bacillus megaterium cy-
tochrome P450 BM3, J. Mol. Catal., B Enzym. 63 (2010) (2010) 179–187, https://
doi.org/10.1016/j.molcatb.2010.01.017.
2
006.07.047.
9] J.J. Nevado, G.C. Peñalvo, R.M. Dorado, V.R. Robledo, Simultaneous determination
of omeprazole and their main metabolites in human urine samples by capillary
electrophoresis using electrospray ionization-mass spectrometry detection, J.
Pharm. Biomed. Anal. 92 (2014) 211–219, https://doi.org/10.1016/j.jpba.2013.
[31] S.H. Park, D.H. Kim, D. Kim, D.H. Kim, H.C. Jung, J.G. Pan, T. Ahn, D. Kim,
C.H. Yun, Engineering bacterial cytochrome P450 (P450) BM3 into a prototype
with human P450 enzyme activity using indigo formation, Drug Metab. Dispos.
1
2.020.
[
10] X.Q. Li, L. Weidolf, R. Simonsson, T.B. Andersson, Enantiomer/enantiomer inter-
actions between the S- and R-isomers of omeprazole in human cytochrome P450
enzymes: major role of CYP2C19 and CYP3A4, J. Pharmacol. Exp. Ther. 315 (2005)
2
010 (May (38)) (2010) 732–739, https://doi.org/10.1124/dmd.109.030759.
[32] J.Y. Kang, S.Y. Kim, D. Kim, D.H. Kim, S.M. Shin, S.H. Park, K.H. Kim, H.C. Jung,
J.G. Pan, Y.H. Joung, Y.T. Chi, H.Z. Chae, T. Ahn, C.H. Yun, Characterization of
diverse natural variants of CYP102A1 found within a species of Bacillus mega-
terium, AMB Express 1 (2011) 1, https://doi.org/10.1186/2191-0855-1-1.
7
77–787, https://doi.org/10.1124/jpet.105.090928.
[
[
11] A.H. Atrakchi, Interpretation and considerations on the safety evaluation of human
drug metabolites, Chem. Res. Toxicol. 22 (2009) 1217–1220, https://doi.org/10.
[
33] E.M. Isin, F.P. Guengerich, Kinetics and thermodynamics of ligand binding by cy-
tochrome P450 3A4, J. Biol. Chem. 281 (2006) 9127–9136, https://doi.org/10.
1
021/tx900124j.
12] S. Schadt, B. Bister, S.K. Chowdhury, C. Funk, C.E.C.A. Hop, W.G. Humphreys,
F. Igarashi, A.D. James, M. Kagan, S.C. Khojasteh, A.N.R. Nedderman, C. Prakash,
F. Runge, H. Scheible, D.K. Spracklin, P. Swart, S. Tse, J. Yuan, R.S. Obach, A
decade in the MIST: learnings from investigations of drug metabolites in drug de-
velopment under the “Metabolites in Safety Testing” regulatory guidance, Drug
Metab. Dispos. 46 (2018) 865–878, https://doi.org/10.1124/dmd.117.079848.
13] J.B. Behrendorff, W. Huang, E.M. Gillam, Directed evolution of cytochrome P450
enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed
substrate specificity, Biochem. J. 467 (2015) 1–15, https://doi.org/10.1042/
BJ20141493.
1
074/jbc.M511375200.
[34] C.D. Sohl, E.M. Isin, R.L. Eoff, G.A. Marsch, D.F. Stec, F.P. Guengerich,
Cooperativity in oxidation reactions catalyzed by cytochrome P450 1A2: highly
cooperative pyrene hydroxylation and multiphasic kinetics of ligand binding, J.
Biol. Chem. 283 (2008) 7293–7308, https://doi.org/10.1074/jbc.M709783200.
[
[
35] Z. Lin, J. Lin, Z. Hu, Z. Cao, 1H and 13C NMR chemical shift assignment for te-
natoprazole, Chin. J. Mag. Res. 26 (2009) 393–399.
[
36] M. El-Badry, E.I. Taha, F.K. Alanazi, I.A. Alsarra, Study of omeprazole stability in
aqueous solution: influence of cyclodextrins, J. Drug Sci. Technol. 19 (2009)
347–351, https://doi.org/10.1016/S1773-2247(09)50072-X.
[
[
14] C.H. Yun, K.H. Kim, D.H. Kim, H.C. Jung, J.G. Pan, The bacterial P450 BM3: a
prototype for a biocatalyst with human P450 activities, Trends Biotechnol. 25
[
37] J. Roberts, M.L. McNaughtan, J. MacLachlan, C. Hunter, O. Pahl, Identification of
the acid-induced degradation products of omeprazole and 5-hydroxyomeprazole by
high-resolution mass spectrometry, Rapid Commun. Mass Spectrom. 32 (2018)
929–941, https://doi.org/10.1002/rcm.8120.
(
2007) 289–298, https://doi.org/10.1016/j.tibtech.2007.05.003.
15] J.Y. Kang, S.H. Ryu, S.H. Park, G.S. Cha, D.H. Kim, K.H. Kim, A.W. Hong, T. Ahn,
J.G. Pan, Y.H. Joung, H.S. Kang, C.H. Yun, Chimeric cytochromes P450 engineered
by domain swapping and random mutagenesis for producing human metabolites of
drugs, Biotechnol. Bioeng. 111 (2014) 1313–1322, https://doi.org/10.1002/bit.
[38] M. Mathew, V.D. Gupta, R.R. Bailey, Stability of omeprazole solutions at various ph
values as determined by high-performance liquid chromatography, Drug Dev. Ind.
Pharm. 21 (1995) 965–971, https://doi.org/10.3109/03639049509026660.
2
5202.
[
39] V. Rea, A.J. Kolkman, E. Vottero, E.J. Stronks, K.A. Ampt, M. Honing,
N.P. Vermeulen, S.S. Wijmenga, J.N. Commandeur, Active site substitution A82W
improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3
mutants as rationalized by spin relaxation nuclear magnetic resonance studies,
Biochemistry 51 (2012) 750–760, https://doi.org/10.1021/bi201433h.
40] B.M. van Vugt-Lussenburg, M.C. Damsten, D.M. Maasdijk, N.P. Vermeulen,
J.N. Commandeur, Heterotropic and homotropic cooperativity by a drug-metabo-
lising mutant of cytochrome P450 BM3, Biochem. Biophys. Res. Commun. 346
(2006) 810–818, https://doi.org/10.1016/j.bbrc.2006.05.179.
[
[
16] C.J. Whitehouse, S.G. Bell, L.L. Wong, P450BM3 (CYP102A1): connecting the dots,
Chem. Soc. Rev. 41 (2012) 1218–1260, https://doi.org/10.1039/c1cs15192d.
17] C.F. Butler, C. Peet, A.E. Mason, M.W. Voice, D. Leys, A.W. Munro, Key mutations
alter the cytochrome P450 BM3 conformational landscape and remove inherent
substrate bias, J. Biol. Chem. 288 (2013) 25387–25399, https://doi.org/10.1074/
jbc.M113.479717.
[
[
[
[
18] C.F. Butler, C. Peet, K.J. McLean, M.T. Baynham, R.T. Blankley, K. Fisher,
S.E. Rigby, D. Leys, M.W. Voice, A.W. Munro, Human P450-like oxidation of diverse
proton pump inhibitor drugs by ‘gatekeeper’ mutants of flavocytochrome P450
BM3, Biochem. J. 460 (2014) 247–259, https://doi.org/10.1042/BJ20140030.
19] S.H. Ryu, B.Y. Park, S.Y. Kim, S.H. Park, H.J. Jung, M. Park, K.D. Park, T. Ahn,
H.S. Kang, C.H. Yun, Regioselective hydroxylation of omeprazole enantiomers by
bacterial CYP102A1 mutants, Drug Metab. Dispos. 42 (2014) 1493–1497, https://
doi.org/10.1124/dmd.114.058636.
[41] P.T. Rajagopalan, S. Lutz, S.J. Benkovic, Coupling interactions of distal residues
enhance dihydrofolate reductase catalysis: mutational effects on hydride transfer
rates, Biochemistry 41 (2002) 12618–12628, https://doi.org/10.1021/bi026369d.
[
42] M.K. Julsing, S. Cornelissen, B. Bühler, A. Schmid, Heme-iron oxygenases: powerful
industrial biocatalysts? Curr. Opin. Chem. Biol. 12 (2008) 177–186, https://doi.
org/10.1016/j.cbpa.2008.01.029.
20] G.E. Tsotsou, A. Sideri, A. Goyal, G. Di Nardo, G. Gilardi, Identification of mutant
Asp251Gly/Gln307His of cytochrome P450 BM3 for the generation of metabolites
of diclofenac, ibuprofen and tolbutamide, Chemistry 18 (2012) 3582–3588, https://
doi.org/10.1002/chem.201102470.
[43] F. Kern, Y. Khatri, M. Litzenburger, R. Bernhardt, CYP267A1 and CYP267B1 from
Sorangium cellulosum so ce56 are highly versatile drug metabolizers, Drug Metab.
Dispos. 44 (2016) 495–504, https://doi.org/10.1124/dmd.115.068486.
9

T.-K. Le, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
[
[
[
44] V.B. Urlacher, M. Girhard, Cytochrome P450 monooxygenases in biotechnology
and synthetic biology, Trends Biotechnol. 37 (2019) 882–897, https://doi.org/10.
(+)‐nootkatone, ChemCatChem 7 (2015) 601–604, https://doi.org/10.1002/cctc.
201402952.
1
016/j.tibtech.2019.01.001.
[47] A. Pennec, F. Hollmann, M.S. Smit, D.J. Opperman, One‐pot conversion of cy-
cloalkanes to Lactones, ChemCatChem 7 (2015) 236–239, https://doi.org/10.
1002/cctc.201402835.
45] A.J. Straathof, S. Panke, A. Schmid, The production of fine chemicals by bio-
transformations, Curr. Opin. Biotechnol. 13 (2002) 548–556, https://doi.org/10.
1
016/S0958-1669(02)00360-9.
[48] A. Dennig, E. Busto, W. Kroutil, K. Faber, Biocatalytic one-pot synthesis of l-
Tyrosine derivatives from Monosubstituted Benzenes, pyruvate, and ammonia, ACS
Catal. 5 (2015) 7503–7506, https://doi.org/10.1021/acscatal.5b02129.
46] S. Schulz, M. Girhard, S.K. Gaßmeyer, V.D. Jäger, D. Schwarze, A. Vogel,
V.B. Urlacher, Selective enzymatic synthesis of the grapefruit flavor
10