Studies on the L-2-hydroxy-acid oxidase 2 catalyzed metabolism of S-mandelic acid and its analogues

Article abstract of DOI:10.1016/j.dmpk.2019.02.003

Mandelic acid (MA) is generally used as a biomarker of the exposure of styrene, which is classified as a class of hazardous environmental pollutants, and also used as an important chiral intermediate in pharmaceutical industry. The previous studies have found the excretion of phenylglyoxylic acid (PGA) in human and rat, a metabolite of MA, was mainly from S-MA rather than R-MA. The metabolic mechanism, however, is not clear. In order to explore the possible metabolic mechanism, the enzyme types involved in the stereoselectivity metabolism of MA were firstly studied, and then human and rat long-chain 2-hydroxy-acid oxidase 2 (HAO2) were recombinantly expressed to study the metabolic profiles of S-MA and its analogues. The results indicated that HAO2 might catalyze the stereoselectivity metabolism of S-MA in rats. Human HAO2 (hHAO2) and rat HAO2 (rHAO2) isozymes β₁ and β₂ were successfully cloned and expressed with high purity and good enzyme activities. The enzyme kinetic profiles of these enzymes were different for S-MA and analogues. The order of catalytic efficiency for hHAO2 and rHAO2, however, was reverse. It might be relevance to the difference in active amino acid residues and loop 4 in human and rat L-2-hydroxy acid oxidase isozyme B crystal structures.

Full text of DOI:10.1016/j.dmpk.2019.02.003

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Studies on the L-2-hydroxy-acid oxidase 2 catalyzed metabolism of S-
mandelic acid and its analogues
Yang Zhang, Chen Su, Jinxiu Lei, Lu Chen, Haihong Hu, Su Zeng, Lushan Yu*
Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China
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a r t i c l e i n f o
a b s t r a c t
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Article history:
Mandelic acid (MA) is generally used as a biomarker of the exposure of styrene, which is classified as a
class of hazardous environmental pollutants, and also used as an important chiral intermediate in
pharmaceutical industry. The previous studies have found the excretion of phenylglyoxylic acid (PGA) in
human and rat, a metabolite of MA, was mainly from S-MA rather than R-MA. The metabolic mechanism,
however, is not clear. In order to explore the possible metabolic mechanism, the enzyme types involved
in the stereoselectivity metabolism of MA were firstly studied, and then human and rat long-chain 2-
hydroxy-acid oxidase 2 (HAO2) were recombinantly expressed to study the metabolic profiles of S-MA
and its analogues. The results indicated that HAO2 might catalyze the stereoselectivity metabolism of S-
MA in rats. Human HAO2 (hHAO2) and rat HAO2 (rHAO2) isozymes b1 and b2 were successfully cloned
and expressed with high purity and good enzyme activities. The enzyme kinetic profiles of these en-
zymes were different for S-MA and analogues. The order of catalytic efficiency for hHAO2 and rHAO2,
however, was reverse. It might be relevance to the difference in active amino acid residues and loop 4 in
human and rat L-2-hydroxy acid oxidase isozyme B crystal structures.
Received 11 November 2018
Received in revised form
7 February 2019
Accepted 14 February 2019
Available online xxx
Keywords:
Mandelic acid
Long-chain 2-hydroxy-acid oxidase 2
Stereoselectivity metabolism
Mandelic acid analogues
Cloning and expression
Species difference
© 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
1. Introduction
administrated S-MA group than that in R-MA group in human and
rat [10,11]. Our previous study indicated that MA can show ster-
Mandelic acid (MA) is a biomarker of the exposure of styrene in
human [1]. Styrene is widely used in plasmatic industry and has
been implicated as reproductive toxicant, neurotoxicant, or
possible carcinogen [2,3]. MA has been clinically used as a urinary
antiseptic, particularly as a bladder irritant during urological pro-
cedures [4]. Lately, MA has gained popularity as a topical skin care
treatment for adult acne. In addition, MA is also used as an
important chiral intermediate in pharmaceutical industry for the
synthesis of cephalosporins, vasodilator cyclandelate, urinary tract
bactericide hexamine mandelate and antispasmodic mandelic acid
benzyl ester [5]. Several dehydrogenases from bacterias, such as
Lactobacillus curvatus [6], Alcaligenes bronchisepticus [7], Pseu-
domonas species [8], Pseudomonas stutzeri SDM [9], exhibit ster-
eoselective oxidation manners for MA and were widely used as
kinases for the chemical synthesis of S-MA and R-MA.
eoselective metabolism in kidney and liver of rats. S-MA can be
metabolized to PGA preferentially. Further study showed that
nonmicrosomal enzymes except alcohol dehydrogenase (ADH) are
responsible for the metabolism of MA in rats [12]. It was also found
that MA could undergo one-directional chiral inversion (S-MA to R-
MA) in Wistar rats by Drummond and collaborators [11]. In order to
explore the enzymes involved in the metabolism of MA in human
and rat, we have recombinantly expressed human alcohol dehy-
drogenase 2 (hADH2) and rat alcohol dehydrogenase 1 (rADH1) and
then studied their catalytic effect to MA. The results indicated that
hADH2 could partly and preferentially transform S-MA into PGA,
whereas rADH1 had no catalytic activities to S-MA or R-MA. In
other words, hADH2 could partly catalyze the stereoselectivity
metabolism of MA. The MA biotransformation shows species dif-
ference between human and rats [13].
The previous studies have found significantly high excretion of
phenylglyoxylic acid (PGA, the metabolite of MA) in oral
Long-chain 2-hydroxy-acid oxidase 2 (HAO2) is an FMN-
dependent enzyme which can catalyze the oxidation of 2-
hydroxy acids into 2-keto acids with oxygen as acceptor. HAO2 is
predominantly expressed in liver and kidney and can oxidize a
series of long-chain aliphatic and aromatic hydroxyacids [14]. Rat
HAO2 (rHAO2) has been also reported to promote the metabolism
* Corresponding author.
E-mail address: yuls@zju.edu.cn (L. Yu).
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https://doi.org/10.1016/j.dmpk.2019.02.003
1347-4367/© 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Zhang Y et al., Studies on the L-2-hydroxy-acid oxidase 2 catalyzed metabolism of S-mandelic acid and its analogues,
Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2019.02.003

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of S-MA [15]. However, it is unclear whether hHAO2 could catalyze
the metabolism of MA. Human HAO2 (hHAO2) was identified by
Jones and collaborators for the first time and its gene sequence is
very similar to that of rHAO2 (74% identity) [16]. There are two
forms of rHAO2, isozymes b1 and b2, identified with the cloning and
sequencing of their cDNAs [15]. The sequence-longer isozyme b2
differs from b1 in only having a three-residue insertion, -VRK-, from
position 188 to 189 in the loop 4 of the mature isozyme b1. The
reported crystal structures of rHAO2 [17,18] clearly showed that
rHAO2 has very similar three-dimensional configurations with an
identical b8a8 barrel fold in comparison with other structurally
characterized family members. On the other hand, the crystal
structure of hHAO2 has not been reported up to now.
In present study, it was first reported of the metabolic profiles,
catalyzed respectively by hHAO2, rHAO2 b1, and rHAO2 b2 recom-
binantly expressed in E. coli, of S-MA and its analogues. We
compared the difference in enzyme kinetics of MA and its ana-
logues catalyzed by human and rat HAO2. This study could help
further characterizing the stereoselective metabolic mechanism of
MA and its analogues in mammals and studying the catalytic
mechanism of human and rat HAO2. The structure of MA analogues
and the reaction of the metabolism of MA to PGA are shown in
Fig. 1.
Ltd. (Taixing, China). S-2-chloromandelic acid (S-2-ClMA), S-methyl
mandelate, phenylglyoxylic acid (PGA), and trifluoroacetic acid
(TFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). S-3-
phenyllactic acid (S-3-PLA), phenylpyruvic acid (PPA), and 2,6-
Dichloroindophenol (DCIP) were purchased from Aladdin
(Shanghai, China). Potassium dihydrogen phosphate, EDTA, and
phosphoric acid were purchased from Sinopharm Chemical Re-
agent (Beijing, China). HPLC grade acetonitrile was purchased from
TEDIA (USA). HPLC grade methanol was purchased from J&K
Chemical Ltd. (Shanghai, China).
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2.2. Enzyme expression and purification
The coding sequence of hHAO2 (PubMed accession: AF231917)
which inserted in the pMAL-c2p expression vector, as described
similarly by Lum [19], was expressed in Rosetta (DE3) Escherichia
coli cells. After an overnight incubation in LB medium at 37 ꢀC, the
cells were diluted 100-fold into rich medium with shaking until the
OD600 values reached 0.5e1.0. IPTG was added into the culture at a
final concentration of 0.5 mmol Lꢁ1 to induce MBP-hHAO2
expression and the incubation continued at 37 ꢀC for an additional
3 h. The cells were harvested by centrifugation at 3000ꢂg for
25 min at 4 ꢀC and the pellet was stored at ꢁ80 ꢀC. The cell pellet
containing MBP-hHAO2 (net weight 5 g) was resuspended in 25 mL
of column buffer (20 mmol Lꢁ1 TriS-HCl pH 7.5, 200 mmol Lꢁ1 NaCl,
1 mmol Lꢁ1 EDTA) and lysed by French Press at 4 ꢀC.
The cell lysate was then centrifuged at 12,000ꢂg for 30 min at
4 ꢀC and the supernatant was transferred to a fresh tube and stored
on ice. Some of supernatant was used for western blot analysis. 50%
slurry amylose resin (New England Biolabs) (3 mL) was equilibrated
with ddH2O and column buffer. The cell lysate-amylose resin
mixture was transferred to an ECONO-PAC column (Bio-Rad Labo-
ratories, CA, USA) and the supernatant was allowed to flow
through. The remaining amylose beads bound with MBP-hHAO2
fusion protein were washed three times with 12 column volumes of
column buffer and the MBP-hHAO2 protein was eluted with 5 ad-
2. Materials and methods
2.1. Chemicals and reagents
pMAL-c2p expression vector was kindly supplied by Institute of
biophysics of Academia sinica. Rosetta (DE3) Escherichia coli was
conserved by our lab. Isopropyl-b-D-thiogalactopyranoside (IPTG)
was purchased from Dingguo-changsheng Biological Technology
Ltd. (Beijing, China). 2-Hydroxyoctanoic acid was purchased from
Alfa Aesar. S-MA was kindly presented by Yiming Fine Chemicals
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ditions of
2 mL elution buffer (column buffer containing
10 mmol Lꢁ1 maltose). The protein was detected by the Bio-Rad
protein assay. Protein concentrations were determined with BCA
method using bovine serum albumin as a standard. Glycerol
(Sigma-Aldrich) was added to a final concentration of 10% and the
solution was mixed well, aliquoted at 200 mL per tube, and stored
at ꢁ80 ꢀC.
Rat HAO2 isozymes b1 and b2 were prepared as described by our
previous method [20]. Briefly, the coding sequence of rat HAO2
(PubMed accession: NM_032082) which inserted in the Pet-28a (þ)
expression vector (Purchased from Beijing Protein Innovation,
Beijing, China) was expressed in Rosetta (DE3) Escherichia coli cells.
IPTG at a final concentration of 1 mmol Lꢁ1 was used to induce HiS-
rHAO2 expression. The column buffer for rHAO2 purification con-
tained 50 mmol Lꢁ1 NaH2PO4 (pH 7.5), 500 mmol Lꢁ1 NaCl, and
10 mmol Lꢁ1 imidazole. Ni-NTA resin (Shanghai Biyuntian Biolog-
ical Co., Ltd., Shanghai, China) was used as stationary phase for
rHAO2 purification. The other steps for rHAO2 purification were
similar to that of hHAO2 purification.
2.3. Western blot analyses
The samples were electrophoresed on 10% gradient gels and
transferred to nitrocellulose using standard techniques. The blot
was blocked in 5% non-fat milk for 1 h and incubated with primary
antibody anti-MBP (1:10,000 dilution) (New England Biolabs),
washed with TBST (TriS-buffered saline containing 0.05% Tween-
20), and incubated with secondary Infrared fluorescence labeled
Goat anti-rabbit antibody (1:15,000 dilution). The signal was
Fig. 1. The structures of MA and its analogues. A: S-mandelic acid (S-MA); B: S-2-
chloromandelic acid (S-2-ClMA); C: S-3-phenyllactic acid (S-3-PLA); D: S-methyl
mandelate; E: The reaction of the metabolism of MA to phenylglyoxylic acid (PGA).
Please cite this article as: Zhang Y et al., Studies on the L-2-hydroxy-acid oxidase 2 catalyzed metabolism of S-mandelic acid and its analogues,
Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2019.02.003

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observed using the Odyssey infrared laser imaging system. The
primary antibodies used for western blot analyses were anti-MBP
(1:10,000 dilution) (New England Biolabs) and anti-HiS-tag
(1:3000 dilution) (Neobioscience Technology Company, Shenzhen,
China) for hHAO2 and rHAO2, respectively.
potassium dihydrogen phosphate (pH 3.5, 20 mmol Lꢁ1)-acetoni-
trile (88:12, v/v), 210 nm for PPA.
All the methods' specificity was good. The linearity was
measured by external standard method. The method was linear
from 2 to 50 m
mol Lꢁ1 for PGA, the lower limit of quantification was
2
m
mol Lꢁ1 (S/N ¼ 10, RSD ¼ 4.7%, n ¼ 5), the RSD of intra-day and
inter-day precisions was ꢃ15%, and the absolute recovery and
relative recovery was 98.1e107.0% and 99.4e106.5%, respectively.
2.4. Recombinant enzymes activities
The calibrate curve ranged from 0.5 to 10 and 2e50 m
mol Lꢁ1 for 2-
9
The activity of recombinant hHAO2 was evaluated by its probe
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ClPGA and PPA, respectively.
substrate 2-hydroxyoctanoic acid. The incubation mixtures (total
volume 200
0.1 mmol Lꢁ1 EDTA, 100
and series of concentration of 2-hydroxyoctanoic acid
(100e2000
mol Lꢁ1). After preincubated for 5 min, the meta-
m
L) containing 0.1 mol Lꢁ1 phosphate buffer (pH 7.5),
m
mol Lꢁ1 DCIP, 0.08 g Lꢁ1 enzyme protein,
2.8. Metabolism data analyses
a
All results are expressed as the mean SD in triplicate experi-
ments. Kinetic parameters including maximum rate of formation
(Vmax) and MichaeliS-Menten constant (apparent Km) were esti-
mated using GraphPad prism software (version 5.01, GraphPad
Software Inc., San Diego, CA) designed for a nonlinear least-square
fit to the standard MichaeliS-Menten equation. Intrinsic clearance
CLint ¼ Vmax/Km. The catalytic constant (kcat) was calculated using
the equation: kcat ¼ Vmax/[E]0, where [E]0 is the total molar con-
centration of enzyme. The student t-test was used for statistical
analysis and statistical significance was defined as P < 0.05.
m
bolism reaction was initiated by adding 2-hydroxyoctanoic acid and
incubated at 30 ꢀC for 1 h. The absorbance at 605 nm was detected
which represented the formation of reduced DCIP. The formation of
reduced DCIP was used to indirectly evaluate the catalytic activity
of hHAO2. The activities of rHAO2 isozymes b1 and b2 were both
good according to our previous reported data [20].
2.5. HAO2 catalyzed metabolism of S-MA and its analogues
The human and rat HAO2 were added to a mixture including
0.1 mol Lꢁ1 phosphate buffer solution (pH 7.5) and 1 mmol Lꢁ1
EDTA. The reaction was started by the addition of S-MA or its an-
3. Results
3.1. Human HAO2 expression and activity assay
alogues to 100 m
mol Lꢁ1 final concentration after preincubating in a
shaking water bath at 37 ꢀC for 5 min. After 2 h incubation, the
reaction was terminated by adding equal volume ice-cold aceto-
nitrile and after centrifugated at 13,000ꢂg for 15 min the super-
natant was injected into HPLC for analysis.
The expression of hHAO2 was detected by western blot analysis
using anti-MBP antibody (Fig. 2A). The results contain miscella-
neous bands, possibly due to the lack of specificity of the antibody.
However, the results showed that there was almost no target pro-
tein band before induction, and the target protein band was
observed after induction and purification. The host plasmid trans-
ferred to the blank plasmid did not contain the target protein band
before and after induction. In conclusion, when induced by IPTG,
hHAO2 expression level showed significantly improved. This shows
that we have successfully obtained hHAO2 with higher purity,
which can be used for further activity studies.
2.6. Kinetics studies
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The kinetic studies were performed using recombinant hHAO2
and recombinant rHAO2 isozymes b1 and b2. The enzymes and
various concentrations S-MA and its analogues were added to
0.1 mol Lꢁ1 phosphate buffer solution (pH 7.5, including 1 mmol Lꢁ1
EDTA), respectively. Concentration ranges for these substrates were
as follows: for human HAO2, 50, 100, 200, 500, 1000, 2000,
The activity of recombinant hHAO2 was assayed using 2-
hydroxyoctanoic acid as substrate by measuring the change of
DCIP in A605 (Fig. 2B). Its Km, Vmax and CLint were
4000
2-ClMA; 10, 20, 40, 80, 160, 320, 500
HAO2, 15, 30, 60, 120, 240, 360
mol Lꢁ1 for S-MA; 5, 10, 20, 40, 80,
160, 320
mol Lꢁ1 for S-2-ClMA; 20, 40, 80, 160, 320, 640,
1000
mol Lꢁ1 for S-3-PLA. The reaction was started by the addition
m
mol Lꢁ1 for S-MA; 10, 20, 40, 80,160, 320, 640
m
mol Lꢁ1 for S-
m
mol Lꢁ1 for S-3-PLA; for rat
0.69 0.07 mmol Lꢁ1, 0.192 0.008 mol minꢁ1$mgꢁ1 protein, and
m
278.3 mL minꢁ1$mgꢁ1 protein, respectively. The results indicated
that the recombinant hHAO2 had good activity. Its Km is similar to
0.735 mmol Lꢁ1 reported [19]. Therefore, in this experiment,
hHAO2 was successfully cloned and expressed, and the obtained
recombinant enzyme has good activity and can be used for further
research. In addition, the recombinant rHAO2 b1 and b2 also
showed good activity according to our previous results [20].
m
m
m
of the diluted enzyme solution after preincubating in a shaking
water bath at 37 ꢀC for 5 min. The reactions were terminated by
adding equal volume ice-cold methanol with 2% TFA for S-MA,
acetonitrile with 1% phosphoric acid for S-2-ClMA and acetonitrile
for S-3-PLA, respectively. The supernatant after centrifugation at
13,000ꢂg for 15 min was detected by HPLC. Data for each substrate
(S-MA, S-2-ClMA, S-3-PLA, respectively) were fitted to MichaeliS-
Menten by Graphpad Prism 5.0, regarding the formation rate of
metabolites (PGA, 2-ClPGA, PPA, respectively) as metabolic rate.
3.2. Substrates studies
We chose several MA analogues to study the effect of the sub-
strates' structures on enzyme kinetics characteristics of the human
and rat HAO2. Human and rat HAO2 could both catalyze these MA
analogues.
2.7. HPLC analysis
Chromatographic separation of sample was performed on an
3.3. Kinetic characterization of human and rat HAO2 with MA
analogues
Agilent Eclipse XDB-C18 column (4.6 mm ꢂ 250 mm, 5
mm, Agilent,
Germany). The flow rate was 1 mL minꢁ1 and the injected volume
was 20
m
L. The mobile phase was potassium dihydrogen phosphate
The kinetic curves of MA analogues catalyzed by the two en-
zymes are fitted to MichaeliS-Menten hyperbolic kinetic profile.
The Eadie-Hofstee plots are straight lines (Fig. 4). The kinetic pa-
rameters are presented in Table 1. The kcat/Km is considered as the
(pH 2.5, 20 mmol Lꢁ1)-methanol (85:15, v/v), the detection wave-
length was 254 nm for PGA; potassium dihydrogen phosphate (pH
2.5, 20 mmol Lꢁ1)-acetonitrile (85:15, v/v), 210 nm for 2-ClPGA;
Please cite this article as: Zhang Y et al., Studies on the L-2-hydroxy-acid oxidase 2 catalyzed metabolism of S-mandelic acid and its analogues,
Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2019.02.003

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Fig. 2. A: Western blot analysis of recombinant human HAO2 expression. 1: Soluble fraction of E. coil Rosetta (DE3) with pMALc2p-hHAO2 before induction; 2: Soluble fraction of E.
coil Rosetta (DE3) with pMALc2p-hHAO2 after induction; 3: Amylose resin-purified fraction of MBP-hHAO2; 4: Soluble fraction of E. coil Rosetta (DE3) with blank pMAL-c2p before
induction; 5: Soluble fraction of E. coil Rosetta (DE3) with blank pMAL-c2p after induction. B: The kinetic curves of 2-hydroxyoctanoic acidcatalyzed by the recombinant human
HAO2 (n ¼ 3). The metabolism of S-MA, S-2-ClMA, and S-3-PLA, but couldn't catalyze the metabolism of S-methyl mandelate (Fig. 3). The results indicated that eCOOH is a
necessary group for HAO2's substrates.
catalytic efficiency index. The enzyme kinetic profiles of these en-
zymes are different for S-MA and analogues. The order of catalytic
efficiency for hHAO2 is S-3-PLA > S-2-ClMA > S-MA. The order of
catalytic efficiency for rHAO2, however, is reverse. For rHAO2, the
Km and kcat values of S-MA were about 2 and 10-fold of that of S-
ClMA, respectively. The kcat/Km value of S-MA was about 5-fold of
that of S-ClMA. These results indicated that rHAO2 b1 and b2 had
significantly higher activities to S-MA than to S-ClMA. The enzyme
kinetics characteristics of rHAO2 b1 and rHAO2 b2 have no big
difference whereas the enzyme kinetics characteristics of hHAO2
and rHAO2 have significant differences.
addition, the experiment also optimized the induction temperature
and induction time, but these conditions have no significant effect
on the expression of protein. Since the activity of the recombinase
can be rapidly identified by the change in the color of the DCIP, this
method is also used to verify the activity of the recombinase. A
detailed discussion can be found in our previous article [20].
DCIP is a universal electron acceptor in this family of enzymes,
and human and rat HAO2 use oxygen as an electron acceptor in the
physiological conditions. In order to better simulate the real situ-
ation in vivo, this experiment used natural oxygen as an electron
acceptor to study the metabolism of human and rat HAO2 on
different structural MA analogues at 37 ꢀC. The results showed that
human and rat HAO2 had significant metabolism to S-MA, S-2-
ClMA and S-3-PLA, but did not metabolize S-methyl mandelate. It
indicated that the binding mode of such enzymes to classical sub-
strates was the hydrogen bonding and electrostatic interaction
between the carboxyl group and the active site arginine and the
adjacent phenylalanine [21].
All enzyme concentrations and incubation times under the
method are the optimal conditions. S-MA is a classical substrate for
rHAO2. It has been reported [17] that there may be two mecha-
nisms in the process of rHAO2 catalyzing S-MA in rats: hydrogen
negative ion transfer mechanism and negative carbon mechanism.
The enzymatic kinetics of human and rat HAO2 on S-MA showed
large species differences. Alignment of the amino acid sequences of
human and rat HAO2 shows that three residues in human HAO2 are
different from active sites in rat HAO2: 78 (A-G), 107 (YeF) and 161
(L-C) and these three amino acid residues have a greater impact on
the hydrogen transfer mechanism [17]. Since the methyl group on
the side chain of ALA at position 78 of rHAO2 can interact with the
benzene ring of S-MA, there is no methyl group at position 78 in
hHAO2, and there is no methyl group in the side chain, thus
weakening the interaction relationship with S-MA. And the steric
hindrance of the 110 amino acid residues may be significantly
related to the structure size of the catalytic substrate. In rHAO2,
corresponding to Tyr at position 107, the hydroxyl group at position
4 of the benzene ring is less than Tyr, and its steric hindrance is
smaller and the active pocket is larger. In addition, in the primary
amino acid sequence of human and rat HAO2, the difference in loop
4 (176e202) is also large, which may result in a large change in the
conformation of loop 4 in the crystal structure of hHAO2. And loop
4 may affect the structure of the amino acid residues at the active
site and the binding to the ligand, thereby affecting the function of
the enzyme.
4. Discussion
In our previous study, it was determined that the tissues in
which S-MA is metabolized are the liver and kidney, and the
metabolic sites are mitochondria and cytoplasm (data not shown).
To further investigate the enzymes that mediate S-MA metabolism,
we searched the ExPASy database based on the similarity of sub-
strate structure, and speculated that the following possible en-
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zymes, including alcohol dehydrogenase (EC 1.1.1.1), L-lactic acid
hydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37),
hydroxypyruvate dehydrogenase (EC 1.1.1.81), 2-hydroxyglutarate
dehydrogenase (EC 1.1.99.2), L-2-hydroxy acid oxidase (EC
1.1.3.15). And our study showed that only MDH and HAO2 inhibitors
inhibited the metabolism of S-MA and S-MA was not metabolized
in hMDH, but whether MDH can mediate S-MA metabolism re-
mains to be considered in further research in view of species dif-
ferences. It has been reported that rHAO2 has a higher catalytic
efficiency for S-MA [15]. Rat HAO2 has high expression in rat kid-
ney, which is consistent with the result of our study, indicating that
rHAO2 may be involved in the metabolism of S-MA in rats. The
results of this experiment showed that cytoplasmic protein has
more metabolism of S-MA than mitochondria, and the inhibitory
effect of HAO inhibitor on mitochondria is stronger than that of
cytoplasmic protein, suggesting that there may be multiple en-
zymes involved in the metabolism of S-MA in cytoplasm.
In the cloning and expression of hHAO2, we initially tried to use
pET-28a (þ) as an expression vector, but there was no expression of
the target protein in either the optimal induction temperature or
the optimized IPTG induction concentration, which was consistent
with the report [16]. Lum and collaborators successfully cloned
hHAO2 in E. coli using the pMAL-c2E vector [19]. Therefore, in this
experiment, pMAL-c2p was selected as an expression vector to
make hHAO2 soluble expression in E. coli. Rare codons are often
considered in prokaryotic expression. Considering the presence of
rare codons, Rosetta (DE3) was chosen as the expression host. In
The difference between the molecular structure of S-2-ClMA
and S-MA is that the 2 positions on the benzene ring are replaced
by chlorine atoms, which changes the electron cloud density of the
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5
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Fig. 3. Chromatograms of different MA analoguesincubated withhuman HAO2. A: S-MA; B: S-2-ClMA; C: S-3-PLA; D: S-Methyl mandelate.
benzene ring, thus affecting the relationship between the benzene
ring and Ala 78 and Ile 161. The molecular structure of S-3-PLA
differs from that of S-MA in that it has one more carbon atom in
the side chain, which increases the steric hindrance and thus affects
the interaction between the benzene ring and the carboxyl oxygen
atom and the amino acid residue of the active site. Therefore, the
catalytic efficiency of rHAO2 for S-2-ClMA and S-3-PLA was lower
than that of S-MA.
The enzymatic kinetic parameters of human and rat HAO2 on S-
MA and analogs were compared. For rHAO2, as the substrate chain
length increases, the catalytic efficiency of the enzyme decreases. In
contrast to rHAO2, hHAO2 has the highest catalytic efficiency for S-
Please cite this article as: Zhang Y et al., Studies on the L-2-hydroxy-acid oxidase 2 catalyzed metabolism of S-mandelic acid and its analogues,
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rHao2(β 1)
rHAO2 (β1)
rHao2(β 2)
rHAO2 (β2)
hHAO2
40
30
20
10
0
1000
1000
800
800
600
400
200
0
40
30
20
10
0
1000
600
800
600
400
400
200
200
0.00
0.05
0.10
mg-1 protein)
0.15
0
V/S(mL· mi n-1
·
9
0
5
10
15
20
0
V/S(mL· mi n-1
·
mg-1 protein)
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0
1000
2000
3000
4000
5000
0
90
180
270
360
450
0
90
180
270
360
450
[S](μmol· L-1
)
[S](μmol· L-1
)
[S](μmol· L-1
)
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A
hHAO2
rHAO2( β 1 )
rHao2(β 2)
hHAO2
rHAO2 (β1)
rHAO2 (β2)
250
200
150
100
50
100
80
60
40
20
0
150
100
50
250
protein)
100
200
150
100
50
80
60
-1
¡¤ mg
-1V(nmol¡¤min
40
0
0
1
2
3
4
5
20
V/S(mL· mi n-1 mg-1 protein)
·
0
0
0
0
3
4
0
200
400
[S](μmol· L-1
600
800
0
100
200 1
2¡¤3m0g-01
400
V/S(mL¡¤min-1
protein)
0
100
200
300
400
)
[S](μmol· L-1
)
[S](μmol· L-1
)
B
hHao2
rHao2(β 1)
rHAO2 (β1)
rHAO2 (β2)
hHAO2
500
300
200
100
0
rHao2(β 2
)
600
400
200
0
400
300
200
100
0
500
400
300
200
100
300
200
100
0
0
0
4
8
12
0
1
2
3
V/S(mL· mi n-1· mg-1 protein)
V/S(mL· mi n-1 mg-1 protein)
·
0
200
[S](μmol· L-1
400
600
0
200
400
600
800
0
400
800
1200
[S](μmol· L-1
)
)
[S](μmol· L-1
)
C
Fig. 4. The kinetic curves of S-MA, S-2-ClMA, and S-3-PLA catalyzed by the recombinant human and rat HAO2
b
1 and
b
2 (n ¼ 3). A. S-MA; B. S-2-ClMA; C. S-3-PLA.
Table 1
Kinetic parameters of different MA analogues in the recombinant human and rat HAO2 (n ¼ 3).
Enzymes
Substrates
Kinetic parameters
[E] (mg$mLꢁ1
)
Km
(
m
M)
Vmax (nmol$minꢁ1
$
CLint (mL$minꢁ1
$
kcat (sꢁ1
)
kcat/Km (mMꢁ1sꢁ1
)
mgꢁ1 protein)
mgꢁ1 protein)
hHAO2
S-MA
0.1
264.6 7.5***
38.7 0.3
0.1
4.9
14.1
18.9
4.6
3.3
23.2
5.1
0.051 0.001***
0.305 0.004*** ###
0.554 0.005
0.596 0.012
0.063 0.001
0.197 0.004
0.669 0.012
0.070 0.002
0.394 0.082
0.2
6.4
18.5
12.7
3.0
2.2
15.6
3.3
* ###
S-2-ClMA
S-3-PLA
S-MA
S-2-ClMA
S-3-PLA
S-MA
0.005
0.02
47.6 2.2
29.9 1.1
47.2 3.1
20.7 0.7#
88.9 5.7###
43.3 2.6
20.5 1.7
127.8 7.8
231.4 2.9
420.9 3.9
893.3 17.4
94.9 0.9
296.1 6.1
1003.0 17.5
104.9 2.4
591.4 11.1
rHAO2(b1
)
)
0.002
0.005
0.004
0.002
0.004
0.004
rHAO2(b2
S-2-ClMA
S-3-PLA
4.6
3.1
MrhHAO2 ¼ 70 kD, MrrHAO2( ¼ MrrHAO2( ¼ 40 kD, 1kD ¼ 40,000 g molꢁ1,CLint ¼ Vmax/Km, kcat(sꢁ1) ¼ Vmax/[E]
.
0
b
1)
b2)
1. Values significantly different from the corresponding value of rHAO2. *, P < 0.05; ***, P < 0.001. 2. Values significantly different from the corresponding value of S-MA.
,
#
P < 0.05; ###, P < 0.001.
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3-PLA, followed by S-2-ClMA, and finally S-MA. This may be due to
the fact that the active pocket of hHAO2 is larger than the rHAO2. In
addition, changes in the conformation of the loop 4 may also be
responsible for this difference. At present, the crystal structure of
hHAO2 has not been reported yet, and its active site and catalytic
mechanism remain to be further studied.
5. Conclusions
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We successfully cloned human and rat HAO2 using prokaryotic
expression system and established its importance in the meta-
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man and rat HAO2 on S-MA structural analogues were also
investigated for the first time. By comparing species differences, it
indicated that the structure of hHAO2 protein was possibly
different from that of rHAO2. The catalytic mechanism of human
and rat HAO2 on S-MA and its analogues needs further research by
computer simulation. In addition, the enzymes involved in the
stereoselective metabolism of MA present in rat hepatic cyto-
plasmic proteins have not been fully elucidated, which requires
further “capture” of the enzyme by protein purification techniques
or by “click chemistry” methods.
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The Authors did not report any conflict of interests.
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^
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Please cite this article as: Zhang Y et al., Studies on the L-2-hydroxy-acid oxidase 2 catalyzed metabolism of S-mandelic acid and its analogues,
Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2019.02.003