A highly efficient galactokinase from Bifidobacterium infantis with broad substrate specificity

Article abstract of DOI:10.1016/j.carres.2012.04.022

Galactokinase (GalK), particularly GalK from Escherichia coli, has been widely employed for the synthesis of sugar-1-phosphates. In this study, a GalK from Bifidobacterium infantis ATCC 15697 (BiGalK) was cloned and over-expressed with a yield of over 80 mg/L cell cultures. The k_cat/K_m value of recombinant BiGalK toward galactose (164 s^-1 mM ^-1) is 296 times higher than that of GalK from E. coli, indicating that BiGalK is much more efficient in the phosphorylation of galactose. The enzyme also exhibits activity toward galacturonic acid, which has never been observed on other wild type GalKs. Further activity assays showed that BiGalK has broad substrate specificity toward both sugars and phosphate donors. These features make BiGalK an attractive candidate for the large scale preparation of galactose-1-phosphate and derivatives.

Full text of DOI:10.1016/j.carres.2012.04.022

Carbohydrate Research 355 (2012) 35–39
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
A highly efficient galactokinase from Bifidobacterium infantis with broad
substrate specificity
⇑
⇑
Lei Li, Yonghui Liu, Wenjun Wang, Jiansong Cheng, Wei Zhao , Peng Wang
College of Pharmacy and State Key Laboratory of Medicinal Chemical Biology, Nankai University, 94 Weijin Road, Nankai District, Tianjin 300071, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Galactokinase (GalK), particularly GalK from Escherichia coli, has been widely employed for the synthesis
of sugar-1-phosphates. In this study, a GalK from Bifidobacterium infantis ATCC 15697 (BiGalK) was cloned
and over-expressed with a yield of over 80 mg/L cell cultures. The kcat/Km value of recombinant BiGalK
toward galactose (164 sꢀ1 mMꢀ1) is 296 times higher than that of GalK from E. coli, indicating that BiGalK
is much more efficient in the phosphorylation of galactose. The enzyme also exhibits activity toward
galacturonic acid, which has never been observed on other wild type GalKs. Further activity assays
showed that BiGalK has broad substrate specificity toward both sugars and phosphate donors. These fea-
tures make BiGalK an attractive candidate for the large scale preparation of galactose-1-phosphate and
derivatives.
Received 20 March 2012
Received in revised form 30 April 2012
Accepted 30 April 2012
Available online 8 May 2012
Keywords:
Bifidobacterium
Galactokinase
Galactose-1-phosphate
Substrate specificity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
GalKs from a variety of organisms including human, yeast,
plants, and bacteria were isolated, expressed, and character-
Galactose (Gal) is a ubiquitous monosaccharide found in glycan
structures of most organisms. The assembly of Gal-containing gly-
cans requires UDP-Gal and galactosyltransferases. Two pathways
were found for the biosynthesis of UDP-Gal: The Leloir pathway
and the salvage pathway (Fig. 1). The galactokinase (EC 2.7.1.6,
ized.5–8 These enzymes exhibit distinct specific activities. For in-
stance, EcGalK has a kcat/Km value of 0.552 sꢀ1 mMꢀ1 9
, whereas
GalK form Saccharomyces cerevisiae (ScGalK) has a kcat/Km value
of 92.7 sꢀ1 mMꢀ1 (Table 1), suggesting that ScGalK is over 150
times more efficient than EcGalK. However, the application of
ScGalK in the synthesis of sugar-1-phosphates was limited by
the low expression level.8 Substrate specificity studies revealed
that GalK could tolerate modifications of Gal at C-2 and C-6. For
example, other than Gal, 2-deoxygalactose (Gal2D), galactosamine
ATP:
D-galactose-1-phosphototransferase, GalK) catalyzed reac-
tion, ATP-dependent phosphorylation of Gal to form galactose-1-
phospahte (Gal-1-P), is shared by both pathways. Over the past five
decades, GalK has attracted significant research attention due to its
important role in sugar metabolism.1,2 Other than the biological
significance, GalKs (particularly GalK from Escherichia coli, EcGalK)
have been extensively investigated and employed for the in vitro
preparation of sugar-1-phosphates for glycorandomization.3,4
(GalN), and 6-deoxygalactose (Gal6D, also known as D-fucose) are
possible substrates of EcGalK9 and ScGalK.10 Recently, structure-
based engineering to further broaden the substrate specificity of
EcGalK greatly improved the in vitro synthesis of natural and
unnatural sugar-1-phosphates. As a milestone, Thorson and co-
workers achieved the preparation of 28 sugar-1-phosphates via
using a novel mutant (Y371H/M173L) of the enzyme.3 Neverthe-
less, large scale synthesis of Gal-1-P and derivatives still partly
suffers from the relative low activity of EcGalK. On the other
hand, in spite of extensive studies on sugar promiscuity, the spec-
ificity of GalK toward phosphate donors has hardly been
explored.11,12
Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; CTP,
cytidine triphosphate; dATP, deoxyadenosine triphosphate; dCTP, deoxycytidine
triphosphate; dGTP, deoxyguanosine triphosphate; dTTP, deoxythymidine triphos-
phate; dUTP, deoxyuridine triphosphate; Gal, galactose; Gal-1-P, galactose-1-
phosphate; Gal2D, 2-deoxygalactose; Gal6D, 6-deoxygalactose; GalA, galacturonic
acid; GalK, galactokinase; GalN, galactosamine; GalNAc, N-acetylgalactosamine;
GDP, guanosine diphosphate; Glc, glucose; GTP, guanosine triphosphate; IPTG,
isopropyl-b-D-1-thiogalactopyranoside; ITP, inosine triphosphate; TLC, thin layer
chromatography; UDP-Gal, uridine 50-diphosphogalactose; UTP, uridine
triphosphate.
In this study, we described the over-expression (80 mg/L cell
culture) and biochemical characterization of a highly efficient GalK
from Bifidobacterium infantis ATCC 15675 (BiGalK). Detailed sub-
strate specificity of the enzyme toward both sugars and phosphate
donors was also investigated.
⇑
Corresponding authors. Tel./fax: +86 22 23505369 (P.W.).
E-mail addresses: wzhao@nankai.edu.cn (W. Zhao), pwang@nankai.edu.cn
(P. Wang).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.04.022

36
L. Li et al. / Carbohydrate Research 355 (2012) 35–39
lected by centrifugation at 4 °C, 6000ꢁg, and re-suspended in buf-
fer A (50 mM Tris–HCl pH 8, 300 mM NaCl, 20 mM imidazole).
After brief sonication on ice, insoluble materials were removed
by centrifugation at 4 °C, 10,000ꢁg for 30 min. Protein purification
was performed by loading the supernatant onto a Ni-NTA agarose
(2 mL) column pre-equilibrated with buffer A. The loaded column
was then washed with 200 mL of buffer A, and the His6-tagged Bi-
GalK was eluted with 10 mL of buffer B (50 mM Tris–HCl pH 7.5,
300 mM NaCl, 250 mM imidazole). After SDS–PAGE analysis, pro-
tein containing fractions were concentrated and desalted using
PD-10 column (GE Life Science, USA) against buffer C (50 mM
Tris–HCl, pH 8.0, 10% glycerol) for long-term storage at 4 °C. The
protein concentration was determined in a 96-well plate via using
Bradford method.
2.4. Enzymatic activity assay
Figure 1. The Leloir pathway (1) and salvage pathway (2) of UDP-Gal biosynthesis.
GalK, galactokinase; GalU, galactose-1-phosphate uridyltransferase; GalE, UDP-Gal
40-epimerase; USP, UDP-sugar pyrophosphorylase.
The kinase activity assay was performed in a reaction system
(100
ll) containing: 8 mM of Gal, 10 mM of ATP, 5 mM of MgCl2,
100 mM of Tris–HCl (pH 8.0), and 12
lg of purified BiGalK. The
2. Materials and methods
2.1. Materials
reaction was incubated at 37 °C for 20 min, then analyzed by
thin-layer chromatography (TLC) using silica gel TLC plates [i-
PrOH/NH4OH/H2O = 7:3:2 (v/v/v)] and the DNS method described
previously9 (The absorbance at 575 nm was recorded by a Varios-
kan Flash Multimode Reader, Thermo Scientific). The compounds
on TLC plates were stained with anisaldehyde/acetic acid/H2SO4/
H2O = 7:3:10:27 (v/v/v/v), and visualized by brief heating.
All chemical reagents unless otherwise stated were purchased
from Sigma. Ni-NTA agarose was from Qiagen. The chromosome
DNA of Bifidobacterium infantis ATCC 15697 was purchased from
American Type Culture Collection. E. coli competent cells
BL21(DE3) and expression vector pET22b were from Novagen Inc.
2.5. Determination of physicochemical properties
2.2. Molecular cloning
Temperature experiments were carried out as described above,
except that 100 ng of BiGalK was used and reactions were incu-
bated in different temperatures (4, 16, 25, 30, 37, 45, 55, 65 °C).
To determine the dependence of BiGalK on metal ions, reactions
were performed at 37 °C with different metal ion salts (MgCl2,
MnCl2, CaCl2, BaCl2, NiSO4) at a concentration of 5 mM. The effect
of ETDA was tested in presence of 5 mM of EDTA. Other conditions
are same as in temperature experiments. pH dependence experi-
ments were performed at 37 °C in 100 mM different buffers (so-
dium acetate, pH 4.0, 5.0, and 6.0; HEPES, pH 7.0; Tris–HCl, pH
7.5, 8.0, and 8.5; glycine–NaOH, pH 9.0, 10.0, and 11.0) in a total
The BiGalK (GeneBank No.: 213690928) gene was amplified by
PCR using B. infantis ATCC 15697 genomic DNA as the template.
Primers with restriction sites underlined were as follows: 2062F:
50-TGACATATGACTGCTGTTGAATTCATTG-30 (Nde I)/2062R: 50-TA-
TAAGCTTGGCCTCGCGGCTCGCGGAAG-30 (Hind III). Yielded DNA
fragments were digested with Nde I and Hind III, followed by liga-
tion with pET22b vector linearized by same restriction enzymes.
The recombinant plasmid was then verified by DNA sequencing
and transformed into E. coli BL21(DE3) for over-expression.
volume of 100 lL, containing 8 mM of Gal, 10 mM of ATP, 5 mM
2.3. Protein over-expression and purification
of MgCl2 and 100 ng of BiGalK. All reactions were allowed to pro-
ceed for 20 min and analyzed by the DNS method.9
E. coli BL21(DE3) cells harboring the aforementioned plasmid
were grown in LB medium at 37 °C till the O.D.600 reached 0.8, fol-
2.6. Determination of kinetic parameters
lowed by the addition of isopropyl-b-D-1-thiogalactopyranoside
(IPTG) to a final concentration of 0.2 mM. Expression was allowed
to proceed for 20 h at 16 °C with rigorous shaking at 220 rpm. Re-
combinant BiGalK was purified by affinity chromatography using
Ni-NTA agarose via standard procedure. Briefly, cells were col-
Reactions were carried out at 37 °C for 5 min in a total volume
of 50 lL in 100 mM Tris–HCl (pH 8.0), containing: ATP, Gal, 5 mM
of MgCl2, and 50 ng of BiGalK. Apparent kinetic parameters were
obtained by varying the concentration of Gal (1, 2, 4, 6, 8 mM) at
Table 1
Kinetics of GalKs from different species
Substrate Enzyme Km
(mM)
Vmax
mol minꢀ1 mgꢀ1
kcat
kcat/Km
Expression level
References
(
l
)
(sꢀ1
)
(sꢀ1 mMꢀ1
)
(mg Lꢀ1
)
Gal
BiGalK
EcGalK
ScGalK
HsGalK
0.895
2.1
0.6
191
1.65
55.8
146
164
>80
25a
1.6b
2
This work
9
5,8
14
1.16
55.8
8.7
0.552
92.7
8.97
0.97
ATP
BiGalK
EcGalK
ScGalK
HsGalK
2.46
2.5
0.15
0.034
191
1.65
55.8
146
1.16
55.8
8.7
59.5
0.464
372
This work
9
5
14
256
a
Unpublished data.
b
1.6 mg ScGalK protein was obtained from 10 g of recombinant yeast cells.

L. Li et al. / Carbohydrate Research 355 (2012) 35–39
37
a fixed concentration of ATP (10 mM), or varying the concentration
of ATP (0.5, 1, 2, 4, 6 mM) at a fixed concentration of Gal (8 mM).
The average value of triplicate assays using the DNS method was
employed to generate Lineweaver–Burk plots, and Km, Vmax, kcat
values were calculated accordingly.
2.7. Determination of specific activities toward sugars and
phosphate donors
Specific activities were determined with 8 mM of various sugars
(Gal, GalN, Gal2D, Gal6D, galacturonic acid (GalA), Glc, or N-acety-
lgalactosamine (GalNAc)) using 10 mM of ATP as phosphate donor,
or with 10 mM of various nucleotides (ATP, dATP, CTP, dCTP, GTP,
dGTP, UTP, dUTP, dTTP, ITP, ADP, or GDP) by fixing Gal at the con-
centration of 8 mM. Other conditions are same as in enzymatic
activity assay, except that varied amounts of BiGalK (30 ng–
Figure 2. SDS–PAGE analysis of BiGalK over-expression and purification. Lanes: (1)
protein standards (molecular weights are indicated in kDa); (2) total cells before
induction; (3) total cells after induction by IPTG; (4) supernatant; (5) flow-through
from Ni-column; (6) elution from Ni-column.
30 lg) were used to make sure the conversion of sugars reached
from 15% to 30%. The average value of triplicate assays using the
DNS method was employed to calculate specific activities. Same
reactions were also carried out at 37 °C for 3 h for TLC analysis.
2.8. Synthesis of Gal-1-P and derivatives
Reaction was performed in a 15 mL tube with 5 mL of 100 mM
Tris–HCl (pH 8.0) containing 44 mM of Gal or derivatives (GalN,
Gal2D, Gal6D, GalA), 40 mM of ATP, 10 mM of MgCl2, and varied
amounts of BiGalK (0.2–2 mg). The reaction mixture was incubated
at 37 °C for 4–24 h till ATP completely converted into ADP (ana-
lyzed by TLC). After quenching by the addition of 5 mL of ice-cold
ethanol, the reaction was kept at 4 °C for 1 h, followed by centrifu-
gation at 4 °C, 6000ꢁg for 30 min to remove insoluble materials.
The supernatant was then concentrated and subjected to P-2
(Bio-rad, USA) gel filtration chromatography (90 ꢁ 2.5 cm) with
50 mM of NH4HCO3 as the mobile phase. Product containing frac-
tions were collected and concentrated under vacuum in 45 °C
water bath. The yielded product was dissolved in 25 mL of distilled
water and concentrated again to remove remanent NH4HCO3. NMR
and HRMS were used to identify the products. NMR spectra were
recorded in D2O with a Bruker AV 400 spectrometer (1H NMR at
400 MHz, 13C NMR at 100 MHz, and 31P at 160 MHz). Chemical
shifts were reported in d (ppm) from an internal standard of TMS
(d 0.00). Coupling constants are reported in hertz. High-resolution
electrospray-ionization mass spectra (HRESIMS) were obtained on
a Varian QFT-ESI mass spectrometer.
Figure 3. TLC analysis of BiGalK activity assay (a) and substrate specificity assays (b
and c). (a) Lanes: (1) Gal; (2) ATP; (3) ADP; (4) Gal-1-P; (5) BiGalK catalyzed
reaction. (b) Sugar substrate specificity, reactions containing: (1) Gal + ATP; (2)
GalN + ATP; (3) GalA + ATP; (4) Gal2D + ATP; (5) Gal6D + ATP. (c) Phosphate donor
specificity, reactions containing: (1) Gal + ATP; (2) Gal + dATP; (3) Gal + CTP; (4)
Gal + dCTP; (5) Gal + GTP; (6) Gal + dGTP; (7) Gal + UTP; (8) Gal + dUTP; (9)
Gal + ITP; (10) Gal + dTTP. Sugar-1-phosphates were indicated by arrows.
SDS–PAGE, consistent with the calculated value. The protein can
be stored at 4 °C in 50 mM Tris–HCl (pH 8.0), 10% glycerol for up
to 4 months without significant loss of activity.
3.2. BiGalK catalyzes the formation of Gal-1-P with high
efficiency
TLC was used to analyze the enzymatic activity of BiGalK. As
shown in Figure 3a, after reaction, the spot on thin layer plate cor-
responding to Gal disappeared (also confirmed by the DNS meth-
od9), while a new spot corresponding to Gal-1-P was detected
(Fig. 3a, lanes 1, 4, 5), accompanying with the conversion of ATP
to ADP (Fig. 3a, lanes 2, 4, 5). HRMS and 1H NMR analysis of the
product further confirmed the generation of Gal-1-P (Supplemen-
tary data). These results demonstrated that BiGalK catalyzes the
phosphorylation of Gal to form Gal-1-P in an ATP-dependent
manner.
3. Results and discussion
3.1. Over-expression and purification of BiGalK
The deduced protein sequence of Blon_2062 from B. infantis
ATCC 15697 shares significant amino acid similarities with EcGalK
(51%) and other characterized GalKs. The protein also contains
characteristic signature motifs of the GHMP superfamily,13 a large
family including galactokinases, homoserine kinases, mevalonate
kinases, and phosphomevalonate kinase. We thus assigned the
protein as a GalK (named BiGalK). To demonstrate its function,
the gene was cloned into pET22b vector, and transformed into
E. coli BL21(DE3) for over-expression. The protein was expressed
with a C-terminal His6-tag under the induction of 0.2 mM IPTG,
mainly in soluble form (Fig. 2, lane 4). It was purified to 95% homo-
geneity (SDS–PAGE analysis) by one-step Ni-NTA affinity chroma-
tography (Fig. 2, lane 6). Typically, over 80 mg of pure BiGalK could
be obtained from 1 L of cell cultures, higher than the expression
levels of EcGalK, ScGalK8, and GalK from Human (HsGalK)14
(Table 1). BiGalK has an apparent molecular weight of 45 kDa on
It has been reported that a GalK isoenzyme isolated from an-
other Bifidobacterium strain (B. bifidum) exhibited high specific
activity toward Gal (318 l
mol/min/mg).6,15 To ascertain whether
our recombinant BiGalK possesses such a high activity, kinetic
studies were performed. As listed in Table 1, BiGalK has Km values
of 0.895 mM and 2.46 mM toward Gal and ATP, respectively,
comparable to that of EcGalK, ScGalK, and HsGalK. The calculated
kcat/Km value (164 sꢀ1 mMꢀ1) toward Gal, however, is 296 times
higher than that of EcGalK (0.552 sꢀ1 mMꢀ1 9
) (Table 1), indicating
that BiGalK is more efficient in the phosphorylation of Gal. Taken
together with the high expression level, BiGalK becomes a more

38
L. Li et al. / Carbohydrate Research 355 (2012) 35–39
Figure 4. Physicochemical properties of BiGalK. (a) pH dependence; (b) temperature dependence; (c) metal ion dependence. All assays were performed in triplicates with the
average yields reported.
attractive potential candidate in the large-scale synthesis of Gal-1-
P and derivatives.
example, they are moderately active with Gal2D and GalN (less
than 10-fold decrease in activity compared to Gal),5,7,9,12 and
weakly active toward Gal6D (over 20-fold decrease in activity com-
pared to Gal).9 Gal derivatives with bulkier groups or modifications
at C-4 position, such as GalA, GalNAc, Glc, are not substrates of
GalK.9 However, GalK from Streptococcus pneumoniae was found
to be an exception, which could use either Glc or GalNAc as
substrate.16
3.3. The physicochemical property of BiGalK
Effects of pH, temperature, and metal ions on BiGalK activity
were investigated subsequently (Fig. 4). BiGalK has an optimum
pH at 8.5, and keeps over 60% of maximal activity at pH between
7.0 and 9.0 (Fig. 4a). Temperature dependence experiments
showed that BiGalK exhibited the highest activity at 55 °C
(Fig. 4b), slightly higher than that of GalKs from B. bifidum
(45 °C),15 Tetrahymena thermophile (41 °C)12, and Streptococcus
pneumoniae (37 °C).16 It is noted BiGalK exhibits over 76% maxi-
mum activity at 65 °C, while only possesses approximately 20%
of maximal activity between 4 °C and 30 °C (Fig. 4b). As expected,
BiGalK requires Mg2+ as cofactors during phosphorylation, like
other enzymes of the GHMP superfamily. Among tested bivalent
metal cations, only Mn2+ can partly substitute Mg2+, with the
reduction of the activity by about 21%.
To investigate whether BiGalK possesses similar sugar substrate
specificity, specific activities toward a series of Gal derivatives were
determined (Table 2). Results showed that BiGalK has high
activity toward Gal (158
l
mol minꢀ1 mgꢀ1) and Gal2D (99.7
l
mol
mol minꢀ1
l
mol minꢀ1 mg
minꢀ1 mgꢀ1), moderate activity toward GalN (12.5
l
mgꢀ1), and weak activity toward Gal6D (0.0781
ꢀ1). Glc is not a substrate of the enzyme, confirming the vital role
of C-4 hydroxyl group in the substrate binding. As expected, GalNAc
is not a substrate of BiGalK because of the bulky C-2 acetamido
group.
Surprisingly, it was found that BiGalK can also catalyze the
phosphorylation of GalA to form GalA-1-phosphate, with a specific
3.4. BiGalK exhibits broad specificity toward sugar substrates
activity of 0.0906 l
mol minꢀ1 mgꢀ1. This is the first wild type GalK
found to possess such an activity. It has been reported that a
Y371H mutation enables EcGalK to accept GalA and 6-amino-gal-
actose,17 possibly due to the reduction of stereo-hindrance effect,
which may enable the accommodation of Gal derivatives with
bulky C-6 groups. Y371 is conserved among GalKs from different
species. Yet in BiGalK the equivalent residue of Y371 is L404, thus
possibly explaining its activity toward GalA.
The formation of Gal-1-P and four derivatives was further con-
firmed by TLC (Fig. 3b), HRMS, and NMR analysis (Supplementary
data).
Substrate specificity studies revealed that GalKs usually could
tolerate modifications of Gal at C-2 and C-6 positions, as long as
modified groups are not bulkier than the hydroxyl group. For
Table 2
Specific activity of BiGalK toward sugars and
phosphate donors
Substrates
Specific activity
mol minꢀ1 mgꢀ1
)
(
l
Gal
158
99.7
12.5
3.5. BiGalK utilizes a variety of nucleoside triphosphates as
phosphate donors
Gal2D
GalN
Gal6D
GalA
Glc
0.0781
0.0906
NDa
Specific activities toward a variety of nucleotides were also
determined to reveal the specificity of BiGalK toward phosphate
donors (Table 2). Interestingly, BiGalK could utilize all of the tested
GalNAc
NDa
nucleoside triphosphates, with ATP (137
l
mol minꢀ1 mgꢀ1) and
ATP
dATP
CTP
dCTP
GTP
dGTP
UTP
dUTP
ITP
dTTP
ADP
GDP
137
138
dATP (138
l
mol minꢀ1 mgꢀ1) as best phosphate donors. In princi-
0.551
0.931
28.5
31.9
0.52
1.50
15.7
2.52
NDa
NDa
ple, nucleoside triphosphates with purine structures (e.g., ATP,
GTP, ITP) serve as better phosphate donors than those with pyrim-
idine structures (e.g., CTP, UTP, dTTP). For example, the specific
activities of BiGalK toward GTP and ITP (28.5
and 15.7
mol minꢀ1 mgꢀ1) are 50 and 27 times higher than that
of CTP (0.551
mol minꢀ1 mgꢀ1), respectively. Similar phenome-
l
mol minꢀ1 mgꢀ1
l
l
non was found as to N-acetylhexosamine 1-kinase from B. longum
JCM1217.18 On the other hand, BiGalK exhibited nearly 12% higher
activity toward dGTP than GTP, and almost twofold higher activi-
a
ND, not detected.

L. Li et al. / Carbohydrate Research 355 (2012) 35–39
39
ties toward dUTP than UTP (Table 2), suggesting that the enzyme
prefers 20-deoxy nucleoside triphosphates. Nucleoside diphos-
phates (e.g., ADP, GDP) are not phosphate donors for BiGalK as
expected.
To further confirm these results, reactions containing Gal and
different nucleoside triphosphates were allowed to proceed for
3 h, followed by TLC analysis. As showed in Figure 3c, Gal-1-P
was generated in all reactions systems with over 50% yield, further
demonstrating that all 10 nucleoside triphosphates serve as suit-
able phosphate donors in the BiGalK-catalyzed reactions.
J3,4 = 2.8 Hz, J2,3 = 10.0 Hz, H-3), 3.77 (dd, 1H, H-2); 13C NMR
(100 MHz, D2O): d 175.8 (C@O), 94.6 (d, J = 6.2 Hz, C-1), 72.3 (C-
5), 70.6 (C-3), 68.3 (C-4), 68.0 (d, J = 7.8 Hz, C-2); 31P NMR
(162 MHz, D2O): d ꢀ0.280 (s). HRESIMS: C6H12O10P (M+H+), calcd
275.0163, found 275.0170.
4. Conclusion
We have identified and biochemically characterized a highly
efficient galactokinase from B. infantis with a kcat/Km value of
164 sꢀ1 mMꢀ1 toward Gal. Over 80 mg of BiGalK could be obtained
from 1 L of cell cultures when over-expressed in E. coli BL21(DE3).
BiGalK is also active toward Gal2D, GalN, and D-Fuc, and most
interestingly, exhibits activity toward GalA, which has never been
found on any other wild type GalKs. Furthermore, the enzyme
could utilize a variety of nucleoside triphosphates as phosphate
donors other than ATP. These features make BiGalK an attractive
candidate for large scale synthesis of sugar-1-phosphates.
3.6. Preparation and characterization of Gal-1-P and derivatives
Gal-1-P and four derivatives were synthesized in 40 mg scale
using purified BiGalK as described in Section 2. Since P-2 gel filtra-
tion using distilled water failed to obtain pure target products,
50 mM of NH4CO3 was tested as the mobile phase. The approach
enabled reliable separation of sugar-1-phosphates and ADP. The
reproducibility of the modified approach was also verified by puri-
fication of mannose-1-phosphate, 2-deoxyglucose-1-phosphate,
N-acetylglucosamine-1-phosphate, and N-acetylgalactosamine-1-
phosphate (unpublished data).
Acknowledgments
This work was supported by Natural Science Foundation of Chi-
na (Grant No. 31100587) and China Postdoctoral Science Founda-
tion (Grant No. 20100480040).
3.6.1. a-D-Galactopyranosyl phosphate (Gal-1-P)
1H NMR (400 MHz, D2O) d 5.40 (dd, J = 7.2, 3.6 Hz, 1H), 4.09–
4.02 (m, 1H), 3.93–3.87 (m, J = 2.8 Hz, 1H), 3.80 (dd, J = 10.2,
3.1 Hz, 1H), 3.71–3.60 (m, 3H). HRESIMS: C6H14O9P (M+H+), calcd
261.0370, found 261.0372.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
3.6.2. 2-Amino-2-deoxy-
1-P)
a-D-galactopyranosyl phosphate (GalN-
j.carres.2012.04.022.
1H NMR (400 MHz, D2O) d 5.58 (dd, J = 7.3, 3.4 Hz, 1H), 4.11 (dd,
J = 7.0, 5.2 Hz, 1H), 4.01 (dd, J = 10.9, 3.2 Hz, 1H), 3.93 (d, J = 3.0 Hz,
1H), 3.68–3.61 (m, 4H), 3.40 (dd, J = 10.8, 2.3 Hz, 1H). HRESIMS:
C6H15NO8P (M+H+), calcd 260.0530, found 260.0535.
References
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3.6.3. 2-Deoxy-a-D-galactopyranosyl phosphate (Gal2D-1-P)
1H NMR (400 MHz, D2O): d 5.55 (dd, J1,P = 7.2 Hz, 1H, H-1), 4.09
(m, 1H, H-5), 4.04 (m, 1H, H-5), 3.82 (dd, 1H, H-4), 3.72–3.63 (m,
2H, H2-6), 1.89 (m, 1H, H-2a), 1.87 (m, 1H, H-2b); 13C NMR
(100 MHz, D2O): d 93.5 (d, J = 4.5 Hz, C-1), 71.6 (C-5), 67.7 (C-4),
64.5 (C-3), 61.7 (C-6), 32.5 (d, J = 6.3 Hz, C-2); 31P NMR
(162 MHz, D2O): d ꢀ0.039 (s). HRESIMS: C6H13O8PNa (M+Na+),
calcd 267.0240, found 267.0238.
3.6.4. 6-Deoxy-a-D-galactopyranosyl phosphate (Gal6D-1-P)
1H NMR (400 MHz, D2O): d 5.37 (dd, J1,2 = 3.2 Hz, J1,P = 14 Hz,
1H, H-1), 4.16 (m, 1H, H-5), 3.87–3.66 (m, 3H, H-2,3,4), 1.12 (d,
3H, H3-6); 13C NMR (100 MHz, D2O): d 94.6 (C-1), 71.8 (C-3), 69.3
(C-5), 68.2 (C-4), 67.2 (d, J = 3.4 Hz, C-2), 15.3 (C-6); 31P NMR
(162 MHz, D2O): d ꢀ0.376 (s). HRESIMS: C6H14O8P (M+H+), calcd
245.0421, found 245.0423.
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3.6.5. a-D-Galactopyranosyluronic acid phosphate (GalA-1-P)
1H NMR (400 MHz, D2O): d 5.49 (dd, J1,2 = 3.6 Hz, J1,P = 6.4 Hz,
1H, H-1), 4.40 (d, 1H, H-5), 4.25 (dd, 1H, H-4), 3.90 (dd,