2422
M. Chen et al. / Carbohydrate Research 346 (2011) 2421–2425
example, in addition to Gal, four sugars (2-deoxy-Gal, 3-deoxy-Gal,
2-amino-2-deoxy-Gal and -fucose) were proved to be substrates
(500 mM NaCl, 20 mM Tris–HCl, pH 8.0, 20 mM imidazole), and
lysed by sonication for 10 min at 400 W. The supernatant was col-
lected by centrifugation (12,000 rpm, 30 min, 4 °C), and loaded
onto a 5-mL HisTrap affinity column which was pre-equilibrated
with binding buffer. The target proteins were eluted with elution
buffer (500 mM NaCl, 20 mM Tris–HCl, pH 8.0, 500 mM imidazole),
and fractions containing the purified enzymes were collected and
dialyzed against Tris–HCl buffer (20 mM, pH 8.0). The purity and
molecular weight of the purified enzyme was analyzed by 12%
SDS–PAGE as described by Laemmli.24 Protein concentration was
determined by the Bradford method.25 The purified enzyme was
named as GalKSpe4 and stored at ꢀ20 °C in 20% glycerol for further
analysis.
D
for GALK from Escherichia coli, while D-Gal and 2-deoxy-Gal were
recognized by human GALKs.10 However, GALK enzymes reported
previously could hardly tolerate modifications at C-4 position.10
Natural anomeric glucokinase capable of D-glucose anomeric phos-
phorylation was only reported in L. lactis GALK with weak activity
(kcat/Km value is 0.7 mMꢀ1 minꢀ1).2 GalNAc could not be utilized by
GALK enzymes in previous reports.2
Streptococcus pneumoniae (the pneumococcus) is a major hu-
man pathogen, which causes more than 1 million deaths each year
worldwide.23 Despite the medical manifestation of the pneumo-
coccus, no reports regarding the GALK in this pathogen were avail-
able. Putative galE, galT, and galK genes involved in the Leloir
pathway (Fig. 1) are present in the S. pneumoniae TIGR4 genome
(GenBank accession No. AE005672), and accordingly, S. pneumoniae
was predicted to be able to utilize exogenous Gal to synthesize Gal-
1-P, yet to be experimentally validated. Characterization of the
pneumococcal GALK would be of great medical importance. Here,
the heterologous expression and biochemical characterization of
the enzyme GALK from S. pneumoniae TIGR4 were described.
2.4. Enzymatic assay and kinetics characterization
The activity of protein GalKSpe4 toward Gal was assayed as de-
scribed before11 with a little modification. Briefly, the reaction
mixture containing Gal (8 mM), ATP (10 mM), MgCl2 (5 mM), and
GALK (6
at 45 °C for 180 min. The reducing sugar consumed in this reaction
was quantified by the DNS method with
-Gal as standard.26 The
lM) in Tris–HCl buffer (20 mM, pH 8.0) was incubated
D
2. Materials and methods
effects of temperature (25–50 °C) and pH (3.0–11.0) on the activity
of GalKSpe4 were determined using Gal as the substrate. Kinetic
data were obtained by determining the slope of linear phase of
the progress curve over 2 min in 30 s intervals and then by calcu-
lating the kinetic parameters as described by Yang et al.12
2.1. Bacterial strains and materials
E. coli DH5a (lacZDM15 hsdR recA) for plasmid maintenance was
purchased from Gibco-BRL (Gaithersburg, MD), and E. coli BL21
(DE3) for protein expression was from Novagen (Carlsbad, CA).
The genomic DNA of S. pneumoniae TIGR4 was kindly provided
by Dr. Samantha King (Nationwide Children’s Hospital™, Colum-
bus, USA). Plasmid pMCSG7 was obtained from Novagen (Madison,
WI). PCR reagents and restriction enzymes were from Invitrogen
(Carlsbad, CA). The plasmid extraction kit and gel purification kit
were purchased from Qiagen (Valencia, CA). The HisTrap affinity
column (5 mL) and HiLoad-16/60-superdex 200 column were from
Amersham Pharmacia Biotech (Piscataway, NJ). All kits and en-
zymes were used following the manufacturer’s instruction.
2.5. Substrate specificity of the enzyme GalKSpe4
The activity of recombinant GalKSpe4 toward Gal, Glc, N-acetyl-
galactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) was
assayed in a cocktail containing 50 mM Tris–HCl (pH 8.0), 5 mM
MgCl2, 5 mM ATP and proper amount of GalKSpe4 at 45 °C for
3 h. The reaction was terminated by boiling the mixture for
5 min followed by centrifugation. The supernatant was analyzed
by normal phase silica gel thin-layer chromatography with the
phase being n-butanol/acetic acid/water (2:1:1, v/v/v).
2.2. Cloning of galK gene from S. pneumoniae TIGR4
2.6. Purification of the phosphorylated products catalyzed by
GalKSpe4
The DNA sequence of galK gene (GenBank accession No.
AAK75925) was extracted from the genome of S. pneumoniae TIGR4
(GenBank accession No. AE005672), and a pair of specific primers
GalKS (50-TACTTCCAATCCAATGCGATGGCACAACATCTTACT-30) and
GalKA (50-TTATCCACTTCCAATGCTAGTCAAGGACGCGAG-30) was
accordingly designed. An 1100-bp DNA fragment was amplified
by polymerase chain reaction (PCR), and then verified by DNA
sequencing. The corrected PCR fragment was cloned into the plas-
mid pMCSG7. The recombinant plasmid pMCSG7-GALK was subse-
The reaction mixture (10 mL), respectively, contained 40 mM
Gal, Glc, GalNAc or GlcNAc, 50 mM ATP, 5 mM MgCl2, and
1.5 mg/mL GalKSpe4 in 100 mM Tris–HCl buffer (pH 8.0). After
incubation at 45 °C for 3 h, the mixture was briefly boiled for
5 min and then centrifuged to remove protein precipitation. The
supernatant was concentrated under reduced pressure and the res-
idue was purified by normal phase silica gel column chromatogra-
phy using gradient CH2Cl2/5 mM NH4HCO3 in methanol as elution
buffer (CH2Cl2/5 mM NH4HCO3 in methanol 1:1 to 0:1). The frac-
tions containing products were collected and concentrated under
reduced pressure.
quently transformed into E. coli DH5a cells. Selected clones were
characterized by restriction mapping and DNA sequencing. The po-
sitive constructs were subsequently transformed into E. coli BL21
(DE3) for protein expression.
2.3. Heterologous expression and enzymatic purification of
GalKSpe4
2.7. Identification of the phosphorylated products catalyzed by
GalKSpe4
E. coli BL21 (DE3) strain harboring the recombinant plasmids
was grown in 1 L of LB medium with shaking at 37 °C, 220 rpm.
When OD600 reached 0.6–0.8, isopropyl-1-thio-b-D-galactospyr-
anoside (IPTG) was added to a final concentration of 0.2 mM for
induction. After expression proceeded overnight at 16 °C, cells
were harvested by centrifugation at 4 °C, and the target proteins
were detected by SDS–PAGE analysis with Coomassie Blue stain-
ing. Then cells were washed, resuspended in the binding buffer
Analytical TLC was carried out on Silica Gel 60 F254 aluminum-
backed plates (E. Merck). The 200–400 mesh size of the same
absorbent was utilized for all chromatographic purifications. All
compounds isolated were sufficiently identified by mass spectra
(MS) and Proton nuclear magnetic resonance (1H NMR) analysis.
MS were recorded on a Bruker MicroTOF spectrometer. 1H NMR
spectra were recorded on a Bruker DPX 400 spectrometer at
400 MHz.