Characterization of glycerol phosphate oxidase from Streptococcus pneumoniae and its application for ketose synthesis

Article abstract of DOI:10.1016/j.bmcl.2014.12.032

Glycerol phosphate oxidase from Streptococcus pneumoniae (GPO_S.pne) was purified and characterized. By the actions of GPO_S.pne and dihydroxyacetone phosphate (DHAP)-dependent aldolases, various ketoses including rare sugars were synthesized with glyceraldehydes as acceptors in a one-pot four-enzyme system.

Full text of DOI:10.1016/j.bmcl.2014.12.032

Bioorganic & Medicinal Chemistry Letters 25 (2015) 504–507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Characterization of glycerol phosphate oxidase from Streptococcus
pneumoniae and its application for ketose synthesis
b,
Zijie Li a,b, Yingxin Qiao b, Li Cai c, Hideki Nakanishi b, , Xiao-Dong Gao
⇑
⇑
a Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
b Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
c Division of Mathematics and Science, University of South Carolina Salkehatchie, Walterboro, SC 29488, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Glycerol phosphate oxidase from Streptococcus pneumoniae (GPOS.pne) was purified and characterized. By
the actions of GPOS.pne and dihydroxyacetone phosphate (DHAP)-dependent aldolases, various ketoses
including rare sugars were synthesized with glyceraldehydes as acceptors in a one-pot four-enzyme
system.
Received 19 September 2014
Revised 9 December 2014
Accepted 11 December 2014
Available online 18 December 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Glycerol phosphate oxidase
Aldolase
Enzymatic synthesis
Rare sugar
Streptococcus pneumoniae
The high stereoselectivity of aldolases in carbon–carbon bond
formation confers them enormous utility as synthetic biocata-
lysts.1–3 Among the aldolase families, dihydroxyacetone phosphate
(DHAP)-dependent aldolases are the most investigated and
exploited, because aldol reactions catalyzed by this group of aldol-
ases can generate two new stereocenters.4,5 Moreover, the stereose-
lectivity of four DHAP-dependent aldolases are complementary,
therefore a set of four possible diastereomers of vicinal diols can
be synthesized.4,6 However, the main disadvantage for this group
of aldolases is their strict specificity for the donor substrate DHAP,
which is a very expensive ($970/250 mg, Sigma–Aldrich) and
unstable chemical.6–8 Therefore, the capability to produce DHAP
from cheap sources could eventually expand the scope of aldolase
reactions.
Although a number of chemical and enzymatic approaches have
been developed for the preparation of DHAP,9 the yield and purity
are not satisfying. Many of the reported enzymatic methods have
not advanced beyond small scale and chemical routes usually suf-
fer from the use of toxic and expensive starting materials.9 In order
to improve the practicality of the aldol reaction by avoiding the use
of DHAP, one of the most efficient approaches is to provide DHAP
in situ from cheaper starting materials. Fessner and co-workers
first established a one-pot system in which DHAP was produced
by glycerol phosphate oxidase (GPO) with -glycerol 3-phosphate
-GP) as the starting material.10 Employing this strategy, we devel-
oped a one-pot four-enzyme approach which can directly produce
rare sugars using an even cheaper starting material DL-glycerol
L
(L
3-phosphate
(DL-GP) instead of
L-glycerol 3-phosphate with
L-rhamnulose-1-phosphate aldolase from Escherichia coli
(RhaDE.coli),
L-fuculose-1-phosphate aldolase from Thermus
thermophilus HB8 (FucAT.HB8
) and D-fructose 1,6-bisphosphate
aldolase from Staphylococcus carnosus (FruAS.car), respectively11–13
(Scheme 1). Since DHAP consumption by aldolases could prevent
product inhibition of GPO by DHAP, aldolase reactions in situ could
also promote the conversion of L-GP into DHAP. Although GPO
from Streptococcus thermophilus (GPOS.the) utilized in our system
is commercially available, the cost is still rather expensive
($196.00/500 UN, Sigma–Aldrich) which impedes the application
in large-scale synthesis.
GPO (EC 1.1.3.21) is a FAD-dependent a-glycerophosphate oxi-
dase14 and the coenzyme FAD can be reoxidized by reacting with
O2 to form H2O2.15 It was reported that GPO belonging to Strepto-
coccus sp. sources is a dimeric16 and cytosolic protein.16,17 Several
GPO enzymes, especially from Streptococcus sp. sources, have been
investigated for their enzymatic activity.10 However, we noticed
that the enzymatic property of GPO from Streptococcus pneumoniae
(GPOS.pne) has not been well studied and exploited in DHAP
generation.
⇑
Corresponding authors. Tel.: +86 510 85910001 (H.N.), +86 510 85197003
(X.D.G.).
E-mail addresses: hideki@jiangnan.edu.cn (H. Nakanishi), xdgao@jiangnan.edu.
cn (X.-D. Gao).
http://dx.doi.org/10.1016/j.bmcl.2014.12.032
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Z. Li et al. / Bioorg. Med. Chem. Lett. 25 (2015) 504–507
505
OH
O
O
2-O3PO
OH
GPO
2-O3PO
H2O2
OH
H
R
DL-glycerol 3-phosphate
DL-GP
O2
DHAP
catalase
aldolase
rt., 22 h
pH = 7.0
H2O
O
OH
O
OH
AP
37 oC, 22 h
pH = 4.7
H2O3PO
HO
R
R
OH
OH
rare sugar/derivatives
GPO = glycerol phosphate oxidase;
AP = acid phosphatase
Scheme 1. One-pot four enzyme synthesis of rare sugars.
Figure 2. The coenzyme FAD is easily dissociable from GPOS.pne
.
Error bars
represent the standard deviation from three parallel experiments.
In order to obtain relatively large amounts of GPO for the
one-pot system, we cloned the glpO gene encoding GPOS.pne and
heterologously overexpressed the target enzyme in Escherichia coli
Rosetta (DE3). The recombinant cells were disrupted by sonication
and purified with Ni2+-NTA column (Fig. 1) (see Supplementary
data for detailed procedures of cloning and purification of
GPOS.pne). The molecular weight of GPOS.pne subunit from SDS–
PAGE was consistent with the theoretical value.18 To our delight,
the expression level of GPOS.pne was very high and the yield
reached about 50 mg per liter cell culture. As racemic glycerol 3-
phosphate (DL-GP) is much cheaper and it has been reported that
determined. The results showed that GPOS.pne exhibited the highest
activity at 30 °C and the relative activity at 50 °C remained more
than 60% of the maximum activity. However, GPOS.pne almost lost
its activity at 60 °C (Fig. 3). Furthermore, GPOS.pne could tolerate a
broad pH range and the optimal pH value was 8.0 (Fig. 4). Consid-
ering the optimal pH values of aldolases are around 7.0 and
GPOS.pne exhibits 85% of the highest activity at pH 7.0, we chose
pH 7.0 to carry out the one-pot reactions.
As a proof-of-concept experiment, GPOS.pne and RhaDE.coli were
firstly employed in the one-pot system with
the acceptor. Consistent with our previous result, the product
-psicose and -sorbose were simultaneously generated and the
ratio ( -psicose/
-sorbose = ꢀ2:1) was determined by ion-exchange
HPLC after calibration with standard curves. After purification by
silica gel chromatography and gel filtration, a mixture of -psicose
and -sorbose was obtained (52% total yield, see Table 1). The
D-glyceraldehyde as
GPO can exclusively oxidize the L-isomer, the activity of GPOS.pne
D
D
was thus firstly examined in the presence of DL-GP and oxygen
in our research. Unexpectedly, compared with the crude enzyme,
the relative activity of pure enzyme was less than 30% (Fig. 2). This
low activity can most likely be attributed to the possibility that
GPOS.pne requires the coenzyme FAD for activity, which could be
easily dissociable during the purification process.19 To confirm this
hypothesis, enough FAD was added into the reaction mixture and
the activity of GPOS.pne was detected again. Consistent with our
expectation, the activity of GPOS.pne was basically recovered to
the same level as the crude enzyme (Fig. 2). Therefore, FAD was
supplemented into the reaction mixture in our subsequent exper-
iments to achieve full GPOS.pne enzymatic activity.
D
D
D
D
mixture containing only two rare sugars could be well separated
by Ca2+ exchange resin chromatography at 70 °C to obtain pure
D
-sorbose and
synthesized with
-isomer (49% yield, see Table 1).
To demonstrate that GPOS.pne is compatible with other DHAP-
D
-psicose. Alternatively, rare sugar
L-fructose was
L
-glyceraldehyde as the acceptor instead of the
D
dependent aldolases, FucAT.HB8 and FruAS.car were also employed
in this one-pot system together with GPOS.pne under the same reac-
tion conditions. Our results showed that when FucAT.HB8 accepting
To reduce the synthetic cost of rare sugars, GPOS.pne pure
enzyme was employed in the one-pot system instead of the
commercial GPOS.the we previously used. To optimize the one-pot
system, firstly the optimal temperature and pH of GPOS.pne were
D
-glyceraldehyde as the acceptor, rare sugar
sized as the dominated product with rare sugar
minor product and the ratio ( -psicose/
D
-psicose was synthe-
D-sorbose as the
D
D
-sorbose = ꢀ9:1) was also
determined by ion-exchange HPLC (70% total yield, see Table 1).
120.00
100.00
80.00
60.00
40.00
20.00
0.00
0
10
20
30
40
50
60
70
Temperature
Figure 1. SDS–PAGE analysis of GPOS.pne expression and purification. Lanes: 1,
protein marker; 2, whole cells not induced; 3, whole cells induced for 16 h; 4,
supernatant of cell lysate; 5, precipitate of cell lysate; 6, purified GPO.
Figure 3. Effect of temperature on the activity of GPOS.pne. Error bars represent the
standard deviation from three parallel experiments.

506
Z. Li et al. / Bioorg. Med. Chem. Lett. 25 (2015) 504–507
120.00
100.00
80.00
60.00
40.00
20.00
0.00
3
4
5
6
7
8
9
10
11
12
pH Value
Phosphate buffer Tris-HCl buffer
Acetate buffer
Glycine buffer
Figure 4. Effect of pH on the activity of GPOS.pne. Error bars represent the standard deviation from three parallel experiments.
Table 1
One-pot four-enzyme synthesis of ketoses with GPOS.pne and aldolases
Enzyme
Acceptor
Product(s)
Yield (%)
52
Ratio
2:1
O
O
OH
O
OH
HO
HO
OH
OH
OH
GPOS.pne, RhaDE.coli
OH OH
OH
OH OH
D-glyceraldehyde
O
D-psicose
D-sorbose
O
OH
HO
HO
OH
OH
OH
OH
GPOS.pne, RhaDE.coli
GPOS.pne, FucAT.HB8
GPOS.pne, FucAT.HB8
49
70
52
1:0
OH
L-fructose
OH
L-glyceraldehyde
O
O
OH
O
OH
HO
HO
OH
OH
OH
OH
9:1
OH OH
OH OH
D-glyceraldehyde
O
D-psicose
OH
D-sorbose
OH
O
O
HO
HO
OH
OH
OH
OH
1.1:1
OH
L-fructose
OH
L-tagatose
OH
OH
L-glyceraldehyde
O
O
OH
OH
OH
GPOS.pne, FruAS.car
56
54
1:0
1:0
OH OH
D-glyceraldehyde
O
D-fructose
O
OH
HO
OH
OH
OH
GPOS.pne, FruAS.car
OH OH
L-glyceraldehyde
L-sorbose
Alternatively, when FucAT.HB8 accepting
acceptor, both rare sugars -fructose and
neously generated (52% total yield) and the ratio was
-tagatose = ꢀ1.1:1 (Table 1), which was consistent with our
previous results.11 Furthermore,
-fructose and -sorbose were
synthesized catalyzed by GPOS.pne and FruAS.car with - and -glyc-
eraldehyde as the acceptor, respectively. After purification by silica
gel chromatography and P-2 gel filtration, the yields for -fructose
and -sorbose were 56% and 54%, respectively (Table 1). With these
results, we are delighted to see that GPOS.pne is very compatible
with aldolase RhaDE.coli, FucAT.HB8 or FruAS.car in the one-pot system.
Moreover, GPOS.pne exhibits comparable activity to the previously
used commercial GPOS.the, because similar synthetic yields were
observed.11–13 However, it is worth noting that GPOS.pne contains
an easily dissociable FAD and displayed optimum activity at
L
-glyceraldehyde as the
-tagatose were simulta-
-fructose/
30 °C while the commercial one contains FAD as a tightly bound
cofactor and is optimally active at 37 °C.
L
L
L
In summary, we successfully expressed GPOS.pne in E. coli heter-
ologously and characterized the enzymatic properties of this
enzyme. Using the one-pot four-enzyme system we developed
before (Scheme 1), a series of ketoses including four rare sugars
were synthesized by replacing the commercial GPOS.the with
GPOS.pne. The scale of the preparative synthesis is about 100 mg
in this study and the reaction system could be easily magnified if
necessary. It demonstrated that GPOS.pne could serve as a promising
alternative to the relatively expensive commercial GPOS.the in large
scale synthesis or fermentation. Considering the useful application
of GPO in generating the expensive substrate DHAP, we believe this
method could contribute to the preparation of more other useful
carbohydrates and derivatives.
L
D
L
D
L
D
L

Z. Li et al. / Bioorg. Med. Chem. Lett. 25 (2015) 504–507
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This work was supported by the National Natural Science
Foundation of China (21302069), Key Laboratory of Industrial Bio-
technology, Ministry of Education, China (KLIB-KF201206), Funda-
mental Research Funds for the Central Universities (JUSRP51319B,
JUSRP311A02, JUSRP1003), China Postdoctoral Science Foundation
(2014M551500), the Key Project of Chinese Ministry of Education
(313027), Agriculture Science Technology Achievement Transfor-
mation Fund (2014GB2A000268) and 111 Project (No. 111-2-06).
L.C. acknowledges the financial support from University of South
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Supplementary data associated with this article can be found, in
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032.
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