Facile enzymatic synthesis of sugar 1-phosphates as substrates for phosphorylases using anomeric kinases

Article abstract of DOI:10.1016/j.carres.2014.10.014

Three sugar 1-phosphates that are donor substrates for phosphorylases were produced at the gram scale from phosphoenolpyruvic acid and the corresponding sugars by the combined action of pyruvate kinase and the corresponding anomeric kinases in good yields. These sugar 1-phosphates were purified through two electrodialysis steps. α-d-Galactose 1-phosphate was finally isolated as crystals of dipotassium salts. α-d-Mannose 1-phosphate and 2-acetamido-2-deoxy-α-d-glucose 1-phosphate were isolated as crystals of bis(cyclohexylammonium) salts.

Full text of DOI:10.1016/j.carres.2014.10.014

Accepted Manuscript
Note
Facile enzymatic synthesis of sugar 1-phosphates as substrates for phosphory-
lases using anomeric kinases
Yuan Liu, Mamoru Nishimoto, Motomitsu Kitaoka
PII:
S0008-6215(14)00376-0
DOI:
http://dx.doi.org/10.1016/j.carres.2014.10.014
CAR 6862
Reference:
Carbohydrate Research
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 July 2014
16 October 2014
17 October 2014
Please cite this article as: Liu, Y., Nishimoto, M., Kitaoka, M., Facile enzymatic synthesis of sugar 1-phosphates
as substrates for phosphorylases using anomeric kinases, Carbohydrate Research (2014), doi: http://dx.doi.org/
10.1016/j.carres.2014.10.014
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Facile enzymatic synthesis of sugar 1-phosphates as substrates for
phosphorylases using anomeric kinases
Yuan Liu, Mamoru Nishimoto, and Motomitsu Kitaoka*
National Food Research Institute, National Agriculture and Food Research
Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Highlights
ꢀ
ꢀ
ꢀ
ꢀ
We describe one-pot enzymatic synthesis of three sugar 1-phosphates.
Anomeric kinases were used in the presence of an ATP-regeneration system.
These sugar 1-phosphates were available as crystals of particular salts.
These sugar 1-phosphates are donor substrates for phosphorylases.
Abbreviations: αGal1P, α-D-galactose 1-phosphate; αGlc1P, α-D-glucose 1-phosphate,
αGlcNAc1P, 2-acetamido-2-deoxy-α-D-glucose 1-phosphate; αMan1P, α-D-mannose
1-phosphate; βGlc1P, β-D-glucose 1-phosphate; GH, glycoside hydrolase; GlcNAc,
2-acetamido-2-deoxy-α-D-glucose; GT, glycosyltransferase; PEP, phosphoenolpyruvic
acid; TLC, thin layer chromatography
*Corresponding author at: National Food Research Institute, National Agriculture and
Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Fax: +81-29-838-7321.
E-mail address: mkitaoka@affrc.go.jp (M. Kitaoka).
1

Abstract
Three sugar 1-phosphates that are donor substrates for phosphorylases were produced at
the gram scale from phosphoenol pyruvic acid and the corresponding sugars by the
combined action of pyruvate kinase and the corresponding anomeric kinases in good
yields. These sugar 1-phosphates were purified through two electrodialysis steps.
α-D-Galactose 1-phosphate was finally isolated as crystals of dipotassium salts.
α-D-Mannose 1-phosphate and 2-acetamido-2-deoxy-α-D-glucose 1-phosphate were
isolated as crystals of bis(cyclohexylammonium) salts.
Key words
sugar 1-phosphate, anomeric kinase, pyruvate kinase, electrodialysis, substrate for
phosphorylase
2

Phosphorylases are a class of enzymes that phosphorolyze glycosyl linkages to
generate sugar 1-phosphates1-4. Because their reactions are reversible and highly
regiospecific, they are expected to be practical catalysts for producing particular
oligosaccharides. Because sugar 1-phosphates are donor substrates for reverse
phosphorolysis, their availability is important for the utilization of phosphorylases.
According to the CAZy classification based on their amino acid sequences,
phosphorylases belong to one of the following families: glycosyltransferase (GT) 4 and
35 and glycoside hydrolase (GH) 13, 65, 94, 112, and 1305. The majority of
phosphorylases phosphorolyze α- or β-D-glucosides to generate α- or β-D-glucose
1-phosphate (αGlc1P or βGlc1P) with anomeric retention or inversion1-4. Preparative
methods of αGlc1P or βGlc1P as pure crystalline forms such as those in salts have been
well documented. These compounds have been generated by phosphorylases from
commercially produced sugar resources. αGlc1P has been reported to be produced from
starch by glycogen phosphorylase as a dipotassium salt6. It has also been produced from
sucrose by sucrose phosphorylase7. βGlc1P has been produced as a
bis(cyclohexylammonium) salt from trehalose by inverting trehalose phosphorylase8.
Several phosphorylases act on glycosides other than glucosides. The GH112
family comprises phosphorlyases that act on β-D-galactosides to generate α-D-galactose
1-phosphate (αGal1P) (1), including 1,3-β-galactosyl-N-acetylhexosamine
phosphorylase (EC 2.4.1.211)9, 10 and 1,4-β-D-galactosyl-L-rhamnose phosphorylase
(EC 2.4.1.247)11. The GH130 family comprises phosphorylases that act on
β-D-mannosides to generate α-D-mannose 1-phosphate (αMan1P) (2), including
1,4-β-mannosyl-glucose phosphorylase (EC 2.4.1.281)12, β-1,4-mannooligosaccharide
phosphorylase (EC 2.4.1.319)13, and 1,4-β-mannosyl-N-acetylglucosamine
phosphorylase (EC 2.4.1.320)14, 15. The GH94 family mainly comprises phosphorylases
that act on β-D-glucosides, although it also includes N,N′-diacetylchitobiose
phosphorylase (EC 2.4.1.280)16 to generate 2-acetamido-2-deoxy-α-D-glucose
1-phosphate (αGlcNAc1P) (3).
Although sugar 1-phosphates can be chemically synthesized17, 18, enzymatic
methods are preferred for their practical synthesis on a large scale. However, it is
difficult to produce sugar 1-phosphates using phosphorylases except for αGlc1P and
βGlc1P because there are no abundantly available resources to be phosphorolyzed by
known enzymes. Although an attempt to change cellobiose phosphorylase into lactose
phosphorylase to obtain αGal1P from lactose has been reported19, the catalytic
efficiency of the mutant enzyme was too low to be used as a practical catalyst.
3

Anomeric kinases are enzymes that phosphorylate the anomeric position of sugars
with the consumption of ATP. The enzyme galactokinase (EC 2.7.1.6) produces αGal1P
from D-galactose (4)20. αMan1P and αGlcNAcP can be produced by
N-acetylhexosamine 1-kinase (EC 2.7.1.162) from D-mannose (5) and
2-acetamido-2-deoxy-D-glucose (GlcNAc) (6), respectively21. However, it is often
noted that kinases are inhibited by high concentrations of ATP, their phosphate donor
substrate22, 23. The substrate inhibition effect restricts the concentration of the substrate
to low values, thus resulting in low production efficiency. To avoid this problem, we
propose to perform the kinase reaction in the presence of an ATP-regenerating system24.
Pyruvate kinase (EC 2.7.1.40) with phosphoenolpyruvic acid (PEP) (7) is one of the
most commonly used ATP-regenerating systems24. This enzyme transfers the phosphate
group of PEP to ADP to generate pyruvic acid (8) and ATP. We confirmed that the
reactions of both galactokinase and N-acetylhexosamine 1-kinase with 1 mM ATP in the
presence of the ATP-regeneration system were much faster than those with 50 mM ATP
(Fig. 1). Here we document the facile synthesis of αGal1P, αGlcNAc1P, and αMan1P
using kinases and the ATP-regenerating system, followed by the isolation of these
compounds by electrodialysis25 and crystallization.
The enzymatic reactions are described in Scheme 1. Each sugar 1-phosphate was
generated with good yields (>90%) from PEP by the combination of pyruvate kinase
and each corresponding anomeric kinase. The resultant reaction mixtures contained
sugar 1-phosphates, pyruvic acid, ATP/ADP, and unreacted monosaccharide. The first
electrodialysis using a 100 Da molecular mass cutoff membrane effectively removed
pyruvic acid from the mixture. After the second electrodialysis with a 300 Da molecular
mass cutoff membrane, sugar 1-phosphates were recovered in the dialysate solution.
The electrodialysis is scale-up ready by increasing the surface area of the membranes
and replaces the chromatographic step often reported for the purification of sugar
1-phosphates. After the purification, the countercations were replaced using a cation
exchange resin, and the sugar 1-phosphates were crystallized as dipotassium salts or
bis(cyclohexylammonium) salts in water/ethanol or water/acetone as pure sugar
1-phosphates.
With the protocol mentioned above, αGal1P, αGlcNAc1P, and αMan1P, which
are donor substrates for phosphorylases, are easily prepared at the gram scale. The facile
production of these compounds will advance the research on phosphorylases by
supplying their substrates.
4

1. Experimental procedure
1.1. Enzymes and chemicals
Pyruvate kinase from rabbit muscle was purchased from Oriental Yeast Co., Ltd.
(Tokyo, Japan). Galactokinase26 and N-acetylhexosamine 1-kinase21 from
Bifidobacterium longum subsp. longum JCM1217 were prepared as described
previously. PEP monosodium salt and ATP disodium salt were purchased from Wako
Pure Chemicals Industry (Osaka, Japan). Other chemicals used were of reagent grade.
1.2. The reactions of anomeric kinases with and without the ATP-regeneration system
The enzymatic reactions were carried out in reaction mixtures (100 µL) at 30 °C.
The composition of each reaction mixture without the ATP-regeneration system was 50
mM ATP (pH adjusted to 7.0 with NaOH), 5 mM MgCl2, 50 mM monosaccharide
(D-galactose and GlcNAc), and anomeric kinase (9.4 µg/mL galactokinase and 100
µg/mL N-acetylhexosamine 1-kinase, respectively). That with the ATP-regeneration
system was 1 mM ATP, 50 mM PEP (pH adjusted to 7.0 with NaOH), 5 mM MgCl2, 50
mM monosaccharide (D-galactose and GlcNAc), anomeric kinases (9.4 µg/mL
galactokinase and 100 µg/mL N-acetylhexosamine 1-kinase, respectively), and 0.42
U/mL pyruvate kinase. After the reactions for 20 h, aliquots (1 µL) of the reaction
mixtures were spotted on a thin layer chromatography (TLC) plate (Kieselgel 60 F254;
Merck, Darmstadt, Germany). The plate was developed with acetonitrile–water (3:1
v/v), soaked in 5% sulfuric acid–methanol solution, and heated in an oven until the
spots of the carbohydrates became sufficiently visible.
1.3. Synthesis of sugar 1-phosphate
Each reaction mixture (100 mL) contained 50 mM PEP, each sugar (51 mM
D-galactose, 51 mM GlcNAc, 60 mM D-mannose), 1.0 mM ATP, 5.0 mM MgCl2, 0.45
U/mL pyruvate kinase, and each anomeric kinase (15 µg/mL galactokinase for
D-galactose, 210 and 105 µg/mL N-acetylhexosamine 1-kinase for D-mannose and
GlcNAc), with the pH adjusted to 7.0 with 1 M NaOH. The reaction mixtures were
incubated at 30 °C for 24 h (D-galactose and GlcNAc) or 72 h (D-mannose). The
reaction was monitored by TLC. After the reaction completed, the reaction mixtures
were heated until boiling in a microwave oven to inactivate the enzymes. After cooling,
the reaction mixtures were centrifuged to remove the precipitated proteins.
1.4. Electrodialysis
Electrodialysis was performed with Microacylizer S1 (Astom Co., Tokyo, Japan).
5

First, the reaction mixture was electrodialyzed through the electrodialysis cartridge
AC-110 (molecular mass cutoff 100 Da, Sunactis, Osaka, Japan) against 100 mL of 1 %
NaNO3 solution until the current was lower than 0.01 A at 3.9 V. At this stage, pyruvic
acid was removed but sugar 1-phosphate remained in the mixture. Then, the mixture
was electrodialyzed through the electrodialysis cartridge AC-220 (molecular mass
cutoff 300 Da, Sunactis) against 30 mL of distilled water until the electrical
conductivity was 0.25 mS/cm. Sugar 1-phophate was recovered in the dialysate.
1.5. αGal1P (1) dipotassium salt
The phosphorylation of D-galactose with galactokinase resulted in the formation
of 48.4 mM αGal1P in 100 mL (total: 4.84 mmol, 97% yield from PEP). After
electrodialysis, 4.37 mmol of αGal1P was recovered. Then, cations were removed from
the dialysate using 10 g of Dowex 50 (strongly acidic cation exchange resin, H+ type,
Wako Pure Chemicals Industry), and the pH was adjusted to 8.5 using 2 M KOH. The
solution was concentrated to 5 mL using a rotary evaporator, followed by dropwise
addition of 35 mL of ethanol to crystalize αGal1P as a dipotassium salt. Finally, 1.25 g
25
1
of αGal1P 2K (3.72 mmol) was obtained. [α]D = +89° (c = 0.50, H2O). H NMR
(D2O): δ 5.47 (dd, 1H, J1,2 3.7, J1,P 7.4 Hz, H-1), 4.17 (dddd, 1H, J5,P 0.5, J4,5 1.2, J5,6b
4.6, J5,6a 7.7 Hz, H-5), 3.97 (dd, 1H, J4,5 1.2, J3,4 3.4 Hz, H-4), 3.89 (dd, 1H, J3,4 1.1, J2,3
10.4 Hz, H-3), 3.76–3.71 (m, 2H, H-2, H-6a), 3.69 (dd, 1H J5,6b 4.6, J6a,6b 11.7 Hz,
H-6b). 13C NMR (D2O): δ 95.5 (d, J1,P 5.3 Hz, C-1), 72.3 (s, C-5), 71.4 (s, C-3), 71.2 (s,
C-4), 70.8 (d, J2,P 6.5 Hz, C-2), 63.1 (s, C-6).
1.6. αMan1P (2) bis(cyclohexylammonium) salt
The phosphorylation of D-mannose with N-acetylhexosamine 1-kinase resulted in
the formation of 46.7 mM αMan1P in 100 mL (total 4.67 mmol, 93% yield from PEP).
After electrodialysis, 4.07 mmol of αMan1P was recovered. Then, cations were
removed from the dialysate using 10 g of Dowex 50 (H+ type), and the pH was adjusted
to 8.5 using cyclohexylamine. The solution was concentrated to 5 mL using a rotary
evaporator, followed by dropwise addition of 40 mL of acetone to crystalize αMan1P as
a bis(cyclohexylammonium) salt. Attempts to crystalize αMan1P as a disodium salt and
dipotassium salt were not successful. Finally, 1.13
g
of αMan1P
25
bis(cyclohexylammonium) salt (2.46 mmol) was obtained. [α]D = +31° (c = 0.50,
H2O). 1H NMR (D2O): δ 5.31 (dd, 1H, J1,2 1.8, J1,P 8.8 Hz, H-1), 3.95–3.92 (m, 2H, H-2,
H-3), 3.90–3.85 (m, 2H, H-5, H-6a), 3.73–3.69 (m, 1H, H-6b), 3.61–3.56 (m, 1H, H-4),
3.17–3.10 (m, 2H, cyclohexylamine H-1), 2.01–1.93 (m, 4H, cyclohexylamine H-2a/6a),
6

1.82–1.75 (m, 4H, cyclohexylamine H-3a/5a), 1.67–1.61 (m, 2H, cyclohexylamine
H-4a), 1.38–1.26 (m, 8H, cyclohexylamine, H-2b/6b, H-3b/5b), 1.20–1.11 (m, 2H,
cyclohexylamine H-4b). 13C NMR (D2O): δ 96.6 (d, J1,P 4.4 Hz, C-1), 74.5 (s, C-5),
72.8 (d, J2,P 6.9 Hz, C-2), 71.8 (s, C-3), 68.8 (s, C-4), 62.9 (s, C-6), 52.1 (s,
cyclohexylamine C-1), 32.0 (s, cyclohexylamine C-2/6), 26.0 (s, cyclohexylamine
C-3/5), 25.5 (s, cyclohexylamine C-4).
1.7. αGlcNAc1P (3) bis(cyclohexylammonium) salt
The phosphorylation of GlcNAc with N-acetylhexosamine 1-kinase resulted in
the formation of 47.3 mM αGlcNAc1P in 100 mL (total: 4.73 mmol, 95% yield from
PEP). After electrodialysis, 4.35 mmol of αGlcNAc1P was recovered. Then, cations
were removed from the dialysate using 10 g of Dowex 50 (H+ type), and the pH was
adjusted to 8.5 using cyclohexylamine. The solution was concentrated to 5 mL using a
rotary evaporator, followed by dropwise addition of 35 mL of acetone to crystalize
αGlcNAc1P as a bis(cyclohexylammonium) salt. Attempts to crystalize αGlcNAc1P as
a disodium salt and dipotassium salt were not successful. Finally, 1.48 g of αGlcNAc1P
25
bis(cyclohexylammonium) salt (2.96 mmol) was obtained. [α]D = +59° (c = 0.53,
1
H2O). H NMR (D2O): δ 5.33 (dd, 1H, J1,2 3.2, J1,P 7.6 Hz, H-1), 3.94 (ddd, 1H, J5,6a 2.2,
J5,6b 5.1, J4,5, 10.1 Hz, H-5), 3.90 (ddd, 1H, J2,P 1.8, J1,2 3.2, J2,3 10.5 Hz, H-2), 3.87 (dd,
1H, J5,6a 2.3, J6a,6b 12.2 Hz, H-6a), 3.78 (dd, 1H, J3,4 9.1, J2,3 10.4 Hz, H-3), 3.76 (dd, 1H,
J5,6b 5.1, J6a,6b 12.2 Hz, H-6b), 3.46 (dd, 1H, J3,4 9.1, J4,5 10.1 Hz, H-4), 3.17–3.10 (m,
2H, cyclohexylamine H-1), 2.04 (s, 3H, Ac), 2.01–1.93 (m, 4H, cyclohexylamine
H-2a/6a), 1.82–1.75 (m, 4H, cyclohexylamine H-3a/5a), 1.67–1.61 (m, 2H,
cyclohexylamine H-4a), 1.38–1.26 (m, 8H, cyclohexylamine, H-2b/6b, H-3b/5b),
13
1.20–1.11 (m, 2H, cyclohexylamine H-4b). C NMR (D2O): δ 176.4 (s, COCH3), 94.4
(d, J1,P 5.1 Hz, C-1), 73.8 (s, C-5), 73.2 (s, C-3), 71.8 (s, C-4), 62.5 (s, C-6), 55.9 (d, J2,P
7.4 Hz, C-2), 52.1 (s, cyclohexylamine C-1), 32.0 (s, cyclohexylamine C-2/6), 26.0 (s,
cyclohexylamine C-3/5), 25.5 (s, cyclohexylamine C-4), 23.8 (s, COCH3).
1.8. Quantification of anions
Sugar 1-phosphates, phosphate, and pyruvic acid were quantified using an ion
chromatography system (ICA-2000, DKK-Toa Corporation, Tokyo, Japan) equipped
with a conductivity detector with a chemical suppressor (ICA-200C, DKK-Toa
Corporation). Anions were separated through a PCI-205 column (DKK-Toa
Corporation) using an aqueous solution containing 1.8 mM Na2CO3 and 1.7 mM
NaHCO3 as the mobile phase at 37 °C with a flow rate of 1.0 mL/min. Sodium cations
7

were removed by the suppressor using 10 mM H2SO4 at a flow rate of 0.65 mL/min.
1.9. Structural analyses
NMR spectra were recorded with Bruker Avance 500 spectrometer (Bruker
Biospin, Rheinstetten, Germany) at 298 K in 99.9 % D2O. t-BuOH (1.23 and 31.3 ppm)
was added to each sample as the internal reference standard for the 1H and 13C NMR
chemical shifts, respectively. Each signal was assigned by analyzing two-dimensional
NMR spectra [double-quantum-filtered correlation spectroscopy (DQF-COSY) and
heteronuclear single-quantum-coherence (HSQC)].
Acknowledgements
This work was supported by Science and Technology Research Promotion
Program for Agriculture, Forestry, Fisheries and Food Industry. The authors thank the
staff of the Instrumental Analysis Center for Food Chemistry of the National Food
Research Institute for recording the NMR spectra. The authors would like to thank
Enago (www.enago.jp) for the English language review.
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9

Figure legend
Fig. 1. TLC analysis of the reactions of anomeric kinases with and without the
ATP-regeneration system.
A, the reaction of galactokinase with D-galactose; B, the reaction of
N-acetylhexosamine 1-kinase with GlcNAc. Lane 1, standard ATP; lanes 2 and 4,
without the ATP-regenerating system (50 mM ATP), t = 0 and 20 h, respectively; lanes 3
and 5, with ATP-regeneration system (1 mM ATP), t = 0 and 20 h, respectively.
10

Scheme 1
11

B
A
D-galactose
GlcNAc
αGal1P
αGlcNAc1P
ATP
ATP
1
2
3
4
5
1
2
3
4
5
Fig. 1
12

Graphical abstract
13

Highlights
ꢀ
ꢀ
ꢀ
ꢀ
We describe one-pot enzymatic synthesis of three sugar 1-phosphates.
Anomeric kinases were used in the presence of an ATP-regeneration system.
These sugar 1-phosphates were available as crystals of particular salts.
These sugar 1-phosphates are donor substrates for phosphorylases.
14