Characterization and mutational analysis of two UDP-galactose 4-epimerases in Streptococcus pneumoniae TIGR4

Article abstract of DOI:10.1134/S0006297918010054

Current clinical treatments for pneumococcal infections have many limitations and are faced with many challenges. New capsular polysaccharide structures must be explored to cope with diseases caused by different serotypes of Streptococcus pneumoniae. UDP-galactose 4-epimerase (GalE) is an essential enzyme involved in polysaccharide synthesis. It is an important virulence factor in many bacterial pathogens. In this study, we found that two genes (galE_sp1 and galE_sp2) are responsible for galactose metabolism in pathogenic S. pneumoniae TIGR4. Both GalE_Sp1 and Gal_ESp2 were shown to catalyze the epimerization of UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal), but only GalE^Sp2 was shown to catalyze the epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc)/UDP-N-acetylgalactosamine (UDP-GalNAc). Interestingly, GalE_Sp2 had 3-fold higher epimerase activity toward UDP-Glc/UDP-Gal than GalE_Sp1. The biochemical properties of GalE_Sp2 were studied. GalE_Sp2 was stable over a wide range of temperatures, between 30 and 70°C, at pH 8.0. The K86G substitution caused GalE_Sp2 to lose its epimerase activity toward UDP-Glc and UDP-Gal; however, substitution C300Y in GalE_Sp2 resulted in only decreased activity toward UDP-GlcNAc and UDP-GalNAc. These results indicate that the Lys86 residue plays a critical role in the activity and substrate specificity of GalE_Sp2.

Full text of DOI:10.1134/S0006297918010054

ISSN 0006ꢀ2979, Biochemistry (Moscow), 2018, Vol. 83, No. 1, pp. 37ꢀ44. © Pleiades Publishing, Ltd., 2018.
Published in Russian in Biokhimiya, 2018, Vol. 83, No. 1, pp. 117ꢀ126.
Characterization and Mutational Analysis of Two UDPꢀGalactose
4
ꢀEpimerases in Streptococcus pneumoniae TIGR4
1,2
1
1
2
2
1
L. L. Chen , D. L. Han , Y. F. Zhai , J. H. Wang , Y. F. Wang , and M. Chen *
1
State Key Laboratory of Microbial Technology, School of Life Sciences,
Shandong University, Jinan, Shandong 250100, China; Eꢀmail: chenmin@sdu.edu.cn
Institute of AgroꢀFood Science and Technology, Shandong Academy of Agricultural Sciences/Key Laboratory
2
of AgroꢀProducts Processing Technology of Shandong Province/Key Laboratory of Novel Food Resources Processing,
Ministry of Agriculture, Jinan 250100, China; Eꢀmail: chenleilei8210@163.com
Received July 5, 2017
Revision received September 30, 2017
Abstract—Current clinical treatments for pneumococcal infections have many limitations and are faced with many chalꢀ
lenges. New capsular polysaccharide structures must be explored to cope with diseases caused by different serotypes of
Streptococcus pneumoniae. UDPꢀgalactose 4ꢀepimerase (GalE) is an essential enzyme involved in polysaccharide synthesis.
It is an important virulence factor in many bacterial pathogens. In this study, we found that two genes (galEsp1 and galEsp2
)
are responsible for galactose metabolism in pathogenic S. pneumoniae TIGR4. Both GalESp1 and GalESp2 were shown to catꢀ
alyze the epimerization of UDPꢀglucose (UDPꢀGlc)/UDPꢀgalactose (UDPꢀGal), but only GalESp2 was shown to catalyze
the epimerization of UDPꢀNꢀacetylglucosamine (UDPꢀGlcNAc)/UDPꢀNꢀacetylgalactosamine (UDPꢀGalNAc).
Interestingly, GalESp2 had 3ꢀfold higher epimerase activity toward UDPꢀGlc/UDPꢀGal than GalESp1. The biochemical
properties of GalESp2 were studied. GalESp2 was stable over a wide range of temperatures, between 30 and 70°C, at pH 8.0.
The K86G substitution caused GalESp2 to lose its epimerase activity toward UDPꢀGlc and UDPꢀGal; however, substitution
C300Y in GalESp2 resulted in only decreased activity toward UDPꢀGlcNAc and UDPꢀGalNAc. These results indicate that
the Lys86 residue plays a critical role in the activity and substrate specificity of GalESp2
.
DOI: 10.1134/S0006297918010054
Keywords: UDPꢀgalactose 4ꢀepimerase (GalE), Streptococcus pneumoniae TIGR4, mutation, substrate specificity
Streptococcus pneumoniae is a notorious human immunogenicity [4]. Understanding the process of CPS
pathogen that can cause various diseases including acute synthesis is important for its potential value in preventing
pneumonia, otitis media, and meningitis [1, 2]. the various diseases caused by S. pneumoniae.
Importantly, the 7ꢀvalent and 13ꢀvalent pneumococcal
The CPS of S. pneumoniae TIGR4 has a linear
conjugate vaccines cannot provide protection against all repeating tetrasaccharide unit that includes an Nꢀacetylꢀ
the pathogenic serotypes of Streptococcus pneumoniae, galactosamine (GalNAc), a phosphorylated galactose, an
such as S. pneumoniae TIGR4. Therefore, it is still urgent acetylated fucose, and an acetylated mannose [5, 6].
that new targets for drug development in preventing During infection and colonization, abundant carbon
pneumococcal diseases should be explored.
sources are needed to support bacterial growth.
Capsular polysaccharide (CPS) protects cells against Biosynthesis of CPS involves a complex pathway that
phagocytosis during infection and is an important viruꢀ includes the uptake of component monosaccharides and
lence factor in many pathogenic bacteria [3]. There are at their conversion to nucleotide derivatives. UDPꢀgalacꢀ
least 90 different types of CPS in S. pneumoniae that are tose 4ꢀepimerase (GalE) is always involved in the biosynꢀ
differentiated by their polysaccharide structure and thesis of capsular or Oꢀantigen polysaccharide units.
GalE is an essential enzyme in the Leloir pathway of
galactose metabolism [7, 8]. It catalyzes the interconverꢀ
sion of UDPꢀgalactose (UDPꢀGal) and UDPꢀglucose
Abbreviations: CPS, capsular polysaccharide; GalE, UDPꢀ
galactose 4ꢀepimerase; UDPꢀGal, UDPꢀgalactose; UDPꢀ
GalNAc, UDPꢀNꢀacetylgalactosamine; UDPꢀGlc, UDPꢀgluꢀ
cose; UDPꢀGlcNAc, UDPꢀNꢀacetylglucosamine.
(
UDPꢀGlc). In many organisms, including S. pneumoniꢀ
ae, this enzyme can also catalyze the interconversion of
*
To whom correspondence should be addressed.
UDPꢀNꢀacetylglucosamine (UDPꢀGlcNAc) and UDPꢀ
37

38
CHEN et al.
Nꢀacetylgalactosamine (UDPꢀGalNAc) [9, 10]. GalE is characterization of GalESp2 was further performed, includꢀ
a key enzyme for the processes of eukaryotic and prokaryꢀ ing analysis of amino acid substitution derivatives, to allow
otic protein glycosylation and the production or secretion for further understanding of S. pneumoniae TIGR4 GalE.
of virulence factors in many bacterial pathogens [11].
Thus, understanding of GalE will be helpful for developꢀ
ing new drugs or vaccine targets and for providing
serotypeꢀindependent protection. GalE isoenzymes from
various organisms, including Escherichia coli, Kluyveroꢀ
MATERIALS AND METHODS
Bacterial strains and materials. The E. coli DH5α
myces fragilis, Trypanosoma brucei, Drosophila used for plasmid maintenance was purchased from
melanogaster, and humans have been characterized [12ꢀ GibcoꢀBRL (USA), and the E. coli BL21(DE3) used for
1
9]. However, little is known about the GalE from S. protein expression was from Novagen (USA). The
pneumoniae, especially regarding its biochemical properꢀ genomic DNA of S. pneumoniae TIGR4 was kindly proꢀ
ties and substrate specificity. vided by Dr. Samantha King (Nationwide Children’s
In this work, the galEsp1 and galEsp2 genes from S. Hospital, Columbus, Ohio, USA). The plasmid
pneumoniae TIGR4 were cloned and heterologously pMCSG7 was provided by Ohio State University (USA).
expressed in E. coli. The substrate specificity of the two Restriction enzymes, DNA polymerases, and T4 ligases
enzymes was characterized. In addition, biochemical were purchased from Fermentas (USA). The HisTrap
Table 1. PCR primers, plasmids, and strains used in this study
Name
Sequence of PCR primers and description of plasmids and strains*
Source
PCR primer
galEsp1ꢀF
5'GGGACCTCCATATGGCAATATTGGTAACAGG (NdeI)
5'CGGGATCCCAATGCGGATCTCAATTATAGC (BamHI)
5'CGCGCCATGGATATGCAAGAAAAGATTTTGGTAA (NcoI)
5'CCGCTCGAGTTTATCACTATTTAGTCTTCAAATC (XhoI)
galEsp1ꢀR
galE ꢀF
sp2
galE ꢀR
sp2
Plasmid
pMCSG7
T7 promoters with MCS, PBR322 replicon, copy number >100,
IPTGꢀinducible, Amp
Novagen, USA
r
r
pMCSG7ꢀgalEsp1
pMCSG7ꢀgalEsp2
pMCSG7ꢀC300Y
pMCSG7ꢀK86G
galEsp1 (NdeI/BamHI), Amp
this study
this study
this study
this study
r
galEsp2 (NcoI/XhoI), Amp
r
pMCSG7, galEsp2 300 Cys mutant (NcoI/XhoI), Amp
r
pMCSG7, galEsp2 86 Lys mutant (NcoI/XhoI), Amp
Strain
E. coli DH5α
FꢀmcrA Δ(mrrꢀhsdRMSꢀmcrBC)ϕ80lacZΔM15 ΔlacX74 recA1 araD139
GibcoꢀBRL, USA
R
Δ(araꢀleu)7697galU galKrpsL (Str ) endA1nupG
–
–
–
E. coli BL21(DE3)
Strain A
F , ompT, hsdSB(rB mB ), gal, dcmrne131 (DE3)
E. coli BL21(DE3) containing pMCSG7ꢀgalEsp1
E. coli BL21(DE3) containing pMCSG7ꢀgalEsp2
E. coli BL21(DE3) containing pMCSG7ꢀgalEsp2 C300Y
E. coli BL21(DE3) containing pMCSG7ꢀgalEsp2 K86G
Novagen, USA
this study
Strain B
this study
Strain C
this study
Strain D
this study
*
Underlined sequences denote recognition sites of the restriction enzymes.
BIOCHEMISTRY (Moscow) Vol. 83 No. 1 2018

MUTATIONAL ANALYSIS OF GalE IN Streptococcus pneumoniae
39
affinity column used in this research was from Amersham SDSꢀPAGE. Protein concentrations were determined
Pharmacia Biotech (USA). The capillary electrophoresis using the Bradford method [20].
equipment was from Beckman Coulter P/ACE MDQ
USA). All kits and enzymes were used according to the and GalESp2 toward UDPꢀGlc, UDPꢀGal, UDPꢀ
manufacturer’s instructions. No endangered or protected GlcNAc, and UDPꢀGalNAc were assayed as described
species were involved in the field studies. before with a minor modification [21]. Briefly, the reacꢀ
Enzymatic assay. The activities of proteins GalESp1
(
Construction of recombinant E. coli strains. The tion mixture consisted of 1 mM substrate (UDPꢀGlc,
DNA sequences of galEsp1 (NP_346261.1) and galEsp2 UDPꢀGal, UDPꢀGlcNAc, or UDPꢀGalNAc) and varyꢀ
(
NP_346051.1) were extracted from the genome of S. ing amounts of freshly prepared enzyme in 20 mM Trisꢀ
pneumoniae TIGR4 (No. AE005672). The primers HCl, pH 8.0, in a total volume of 50 μl. The reactions
galE ꢀF/R and galE ꢀF/R were designed accordingly were performed at 37°C for 120 min and then quenched
sp1
sp2
and are listed in Table 1. DNA fragments were amplified by boiling for 5 min before centrifugation. The superꢀ
using PCR. The PCR products and plasmid pMCSG7 natant was analyzed using capillary electrophoresis (see
were digested with the same restriction enzymes (Table 1) Supplement to this paper on the site of the journal
and ligated with T4 ligases. Vectors pMCSG7ꢀgalEsp1 and http://protein.bio.msu.ru/biokhimiya and Springer site
pMCSG7ꢀgalEsp2 were constructed and verified by DNA Link.springer.com). The conditions used were as follows:
sequencing. The recombinant plasmids pMCSG7ꢀgalEsp1 75 μm, 25 kV/80 μA, 5ꢀs vacuum injection, monitoring at
and pMCSG7ꢀgalEsp2 were subsequently transformed 262 nm, a 25ꢀmM sodium tetraborate running buffer, pH
into E. coli BL21(DE3) cells for protein expression.
9.4. The activities of enzymes GalESp1 and GalESp2
Sequence alignment and mutagenesis. The sequences toward UDPꢀGal, UDPꢀGlcNAc, and UDPꢀGalNAc
of GalESp1 and GalESp2 and those of six other GalE were assessed using the same method.
sequences belonging to different groups were aligned
using the DNAMAN software. For GalESp2, four other
sequences of GalEs were chosen from group 2 and aligned
to identify the essential sites for the isomerase activity.
According to differences in their sequences and hypoꢀ
RESULTS
Sequence analysis of GalESp1 and GalESp2 from S.
thetical crystal structure, amino acid substitution mutaꢀ pneumoniae TIGR4. The genes of GalESp1 and GalESp2
tions were produced in GalESp2 using the QuickꢀChange cloned from the genome of S. pneumoniae TIGR4 have
siteꢀdirected mutagenesis kit from Stratagene (USA). sizes of 1011 and 1020 bp, respectively. Sequence analysis
Mutants were constructed in the highly conserved active showed high similarity (52.22%) between eight UDPꢀ
residues of Cys300 and Lys86 in GalESp2 using pMCSG7ꢀ hexose 4ꢀepimerases’ amino acid sequences (Fig. 1).
galEsp2 as a template. The mutated plasmids pMCSG7ꢀ
C300Y and pMCSG7ꢀK86G were transformed into E. ly of shortꢀchain dehydrogenase/reductase (SDR) having
coli BL21 for protein expression. twoꢀdomain structure. The Nꢀterminal domain with conꢀ
UDPꢀhexose 4ꢀepimerases belong to the superfamiꢀ
Heterologous expression and enzymatic purification served sequence GxxGxxG forms a modified Rossmann
+
of GalESp1 and GalESp2. The E. coli BL21(DE3) strain fold and is involved in binding of the cofactor NAD ,
harboring the recombinant plasmids was grown in 1 liter whereas a smaller domain with conserved sequence
of Luria–Bertani (LB) broth with shaking at 220 rpm and YxxxK is involved in substrate binding [8]. Both functionꢀ
3
7°C. When the OD600 reached 0.6ꢀ0.8, isopropylꢀ1ꢀthioꢀ al motifs conserved in the SDR superfamily members
βꢀDꢀgalactopyranoside (IPTG) was added to final conꢀ were identified in GalESp1 and GalESp2 (Fig. 1).
centration 0.2 mM for induction. Expression proceeded Based on its substrate specificity, GalEs can be dividꢀ
overnight at 16°C, and then cells were harvested by cenꢀ ed into three groups. Group 1 epimerases strongly prefer
trifugation at 4°C. The cells were washed and resuspendꢀ nonꢀacetylated substrates (UDPꢀGlc/Gal), such as the
ed in 40 ml of start buffer (500 mM NaCl, 20 mM Trisꢀ E. coli GalE, with a corresponding “Y300” residue.
HCl buffer, pH 8.0) and lysed on ice by sonication for Group 2 epimerases can epimerize both acetylated
2
4
0 min at 400 W. The lysate was centrifuged at 12,000g at (UDPꢀGlcNAc/GalNAc) and nonꢀacetylated substrates,
°C for 30 min, and the supernatant was loaded onto a such as the GalE from Drosophila melanogaster. Group 3
HisTrap affinity column (5 ml) preequilibrated with a epimerases include WbgU from Pl. shigelloides [22] and
binding buffer. After washing with 10 column volumes of WbpP from Ps. aeruginosa [23] and show a strong preferꢀ
washing buffer (500 mM NaCl, 20 mM TrisꢀHCl buffer, ence for acetylated substrates with a corresponding
2
0 mM imidazole, pH 8.0), the target Hisꢀtagged proteins “G86” residue.
were eluted with elution buffer (500 mM NaCl, 20 mM According to Ishiyama et al. [23], there are six key
TrisꢀHCl buffer, 500 mM imidazole, pH 8.0). Fractions residues (marked in Fig. 1) related to the group identifiꢀ
containing the purified enzymes were harvested and cation. GalE enzymes belonging to group 1 contain “Lꢀ
desalted with 50 mM TrisꢀHCl buffer (pH 7.5) at 4°C. SꢀYꢀNꢀHꢀL or KꢀSꢀYꢀNꢀNꢀY” in the amino acid
The purity and molecular mass were analyzed using 12% sequences, enzymes belonging to group 2 contain “KꢀSꢀ
BIOCHEMISTRY (Moscow) Vol. 83 No. 1 2018

40
CHEN et al.
Fig. 1. Sequence alignments of GalE from different organisms. The protein sequences are as follows: GalESp1 (WP_000996560.1, group 2),
GalESp2 (WP_001156518.1, group 2), GalEhu from Homo sapiens (NP_001008217.1, group 2), GneYen from Yersinia enterocolitica
(
WP_005167749.1, group 2), GneBsu from Bacillus (WP_003244356.1, group 2), WbpPPae from Pseudomonas aeruginosa (AAF23998.1,
group 3), WbgUPsh from Plesiomonas shigelloides (AAG17409.1, group 3), and GalEEco from Escherichia coli (EFJ65355.1, group 1).
Identical and similar amino acids are shaded in black and gray, respectively. Sequence numbering corresponding to each protein is shown on
the right. The key six residues related to the group identification including the mutation sites K86 and C300 are indicated by stars. The conꢀ
served NADꢀbinding motif (GxxGxxG), the substrateꢀbinding motif (YxxxK), and catalytic amino acid residues responsible for proton transꢀ
fer are boxed, respectively.
YꢀNꢀNꢀC”, and enzymes belonging to group 3 contain (pH 8.0). GalESp1 and GalESp2 were shown to be purified
GꢀSꢀYꢀNꢀAꢀS”. Based on the rules, both the GalEsp1 to homogeneity with apparent molecular mass of about
“
and GalEsp2 sequences from S. pneumoniae TIGR4 were 38 kDa, which is consistent with the molecular masses
more consistent with those of the group 2 epimerases. calculated from their amino acid sequences.
Moreover, it was expected that changing C (group 2) to Y
(
(
group 1) or C (group 2) to S (group 3), as well as K
group 2) to G (group 3), might change the substrate
Table 2. Activity of GalESp1 and GalESp2 enzymes
Enzyme
Substrate
Product
Activity,
mU/mg
specificity of the epimerases.
Heterologous expression and purification of enzymes
GalESp1 and GalESp2. To identify the enzymes GalESp1 and
GalESp2, their encoding genes were cloned into plasmid
pMCSG7. The recombinant enzymes were expressed in
E. coli and purified by nickelꢀaffinity chromatography
GalESp1 UDPꢀGlc
UDPꢀGal
5.4
0
UDPꢀGlcNAc UDPꢀGalNAc
GalESp2 UDPꢀGlc
UDPꢀGal
18.3
18.5
(
Fig. 2). Fractions containing the purified enzymes were
UDPꢀGlcNAc UDPꢀGalNAc
collected and dialyzed against 20 mM TrisꢀHCl buffer
BIOCHEMISTRY (Moscow) Vol. 83 No. 1 2018

MUTATIONAL ANALYSIS OF GalE IN Streptococcus pneumoniae
41
kDa
16.0
M
1
2
3
4
Substrate specificity of GalESp1 and GalESp2. The
reaction supernatant was analyzed by capillary elecꢀ
trophoresis after the enzymatic assay. Both enzymes were
used in an activity assay with UDPꢀGlc and UDPꢀ
GlcNAc using the methods described above. GalESp1
could use only UDPꢀGlc and UDPꢀGal as its substrate,
and its conversion ratios were 30 and 10%, respectively.
GalESp2 could convert both UDPꢀGlc/UDPꢀGal and
UDPꢀGlcNAc/UDPꢀGalNAc (Fig. 3) with conversion
ratios of 29 and 28% for the UDPꢀGlc and UDPꢀ
GlcNAc substrates. The experimental results suggest that
GalESp2 belongs to group 2 as predicted; however, they
also suggest that GalESp1 belongs to group 1 according to
its substrate specificity.
1
66.2
4
5.0
5.0
3
25.0
1
8.4
4.4
The activity of GalESp2 (18.3 mU/mg) was approxiꢀ
mately 3ꢀfold higher than that of GalESp1 (5.4 mU/mg)
when UDPꢀGlc/UDPꢀGal was used as the substrate
1
(
Table 2).
Optimum temperature assay of GalESp2. To further
Fig. 2. SDSꢀPAGE of GalESp1 and GalESp2. Lanes: M) protein
standards; 1, 2) GalESp1 samples eluted by the elution buffer; 3, 4)
GalESp2 samples eluted by the elution buffer.
characterize GalESp2, the optimum temperature of
AU
AU
a
b
0
.010
0.009
.008
0.007
.006
.005
.004
0
.012
0
0
0
0
0
0
0
0
0
0
0
0
.011
.010
.009
.008
.007
.006
.005
.004
.003
.002
.001
.000
0
UDPꢀGlc
UDPꢀGlc
4.34
4.25
0
0
0
UDPꢀGal
UDPꢀGal
0.003
.002
0.001
4
.59
4.50
0
6.04
6.20
6.60
4.70
0
.000
2
.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
2.25 2.75 3.25 3.75 4.25 4.75 5.25 5.75 6.25 6.75
2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
2
.75 3.25 3.75 4.25 4.75 5.25 5.75 6.25 6.75 7.25 7.75
AU
AU
c
0.011
d
0.010
0.009
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0.000
UDPꢀGlcNAc
0.010
0.009
0.008
0.007
0.006
4.07
UDPꢀGlcNAc
4.12
0
0
.005
.004
UDPꢀGalNAc
4.40
0.003
5.48
0.002
4.98
6.03
6.59
6.6
4.35
4.23 4.51
4.37 5.63
0.001
4.67
4.7
3
.63
4.24
4.58
4.5
0.000
3
.4 3.8 4.2
4.6
5.0 5.4 5.8 6.2
5.2 5.6 6.0 6.4
7.0
3.3
3.5
3.7
3.9
4.1
4.3
4.9
3.6 4.0
4.4 4.8
6.8 7.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
Minutes
Fig. 3. Activities of GalESp1 and GalESp2 toward UDPꢀGlc and UDPꢀGlcNAc. a) Activity of GalESp1 toward UDPꢀGlc; b) activity of GalESp2
toward UDPꢀGlc; c) activity of GalESp1 toward UDPꢀGlcNAc; d) activity of GalESp2 toward UDPꢀGlcNAc. The activity of GalESp1/GalESp2
toward Gal is similar to the activity toward Glc. Similarly, the activity of GalESp1/GalESp2 toward UDPꢀGalNAc is similar to their activity
toward GlcNAc.
BIOCHEMISTRY (Moscow) Vol. 83 No. 1 2018

42
CHEN et al.
GalESp2 was explored in 20 mM TrisꢀHCl, pH 8.0, with a
1
80
60
total volume of 50 μl (with 2 μg/ml enzyme and 0.5 mM
UDPꢀGlc). Activities were checked between 30 and 70°C
in 10°C increments as well as at 37°C for 5 min. We found
that GalESp2 still had strong activity even at 70°C (Fig. 4).
Mutation construction and enzymatic assay of
1
140
120
100
GalESp2. To find the functional amino acids of GalESp2
mutational studies were completed on the conserved catꢀ
alytic motif.
,
8
0
60
4
0
0
In this study, specific residues Cys300 and Lys86,
which are highly conserved and supposed to be determined
based on the substrate specificity of GalESp2, were mutatꢀ
ed. Substitution C300Y was expected shift the epimerase
from a group 2 to group 1 sequence signature, and substiꢀ
tution K86G was expected shift the enzyme from a group
2
0
30
37
40
50
60
70
Temperature, °C
2
to group 3 sequence signature. Therefore, substitutions
Fig. 4. Effects of temperature on the activity of GalESp2. The
activity of GalESp2 was determined by measuring the formation of
UDPꢀGal from UDPꢀGlc at 37°C at pH 8.0, which was defined
as 100%.
C300Y and K86G were constructed in GalESp2
The activities of the mutants were analyzed in the
same way as for the native GalESp2 using capillary elecꢀ
.
AU
a
b
AU
0
.009
.008
0.011
0.010
.009
.008
.007
.006
.005
.004
0.003
UDPꢀGlc
UDPꢀGlcNAc
0
4.24
4.03
0
0
0
0
0
0
0
0
0
.007
.006
.005
0
0
0
0
0
.004
.003
.002
.001
.000
UDPꢀGalNAc
4
.45
0
0
0
.002
.001
.000
4
.14
5.39
4
.10
5.45
4
.15
5.60
–0.001
3
.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8
3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9 5.1 5.3 5.5 5.7
3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6
3
.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9 5.1 5.3 5.5 5.7
AU
AU
c
0.013
d
0
0
0
.010
.009
.008
0
.012
0.011
0
0
.010
.009
UDPꢀGlcNAc
UDPꢀGlc
3.99
0
0
0
.007
.006
.005
4.18
0.008
0.007
0
0
.006
.005
UDPꢀGalNAc
5
.45
0
0
0
.004
.003
.002
UDPꢀGal
.41
0.004
0.003
4.38
4.25
4
4.89
0
0
.002
.001
0.000
0.001
5.82
5.8
4
.13
6.34
5.32
5.4
5
.84
0
0
.001
.000
–
2
.6 3.0
3.4
3.2
3.8 4.2
3.6 4.0
4.6 5.0
5.4
5.8 6.2
6.0
3.0
3.4
3.8
3.6
4.2
4.6
5.0
5.2
6.2
2.8
4.4
4.8 5.2 5.6
6.4
3.2
4.0
4.4
4.8
5.6
6.0
6.4
Minutes
Fig. 5. Activity of GalESp2 with substitutions K86G and C300Y toward UDPꢀGlc and UDPꢀGlcNAc. a) Activity of GalESp2 with substitution
K86G toward UDPꢀGlc; b) activity of GalESp2 with substitution K86G toward UDPꢀGlcNAc; c) activity of GalESp2 with substitution C300Y
toward UDPꢀGlc; d) activity of GalESp2 with substitution C300Y toward UDPꢀGlcNAc. The activity of the enzymes with substitutions K86G
and C300Y toward Gal is similar to the activity toward UDPꢀGlc, and the activity of the enzymes with substitutions K86G and C300Y toward
UDPꢀGalNAc is similar to the activity toward UDPꢀGlcNAc.
BIOCHEMISTRY (Moscow) Vol. 83 No. 1 2018

MUTATIONAL ANALYSIS OF GalE IN Streptococcus pneumoniae
43
trophoresis. Substitution C300Y did not change the of the enzyme and the cofactorꢀbinding pocket. The other
GalESp2 substrate character specificity, but it greatly is YxxxK, which is involved in catalysis, in which Y and K
reduced its activity toward UDPꢀGlcNAc/UDPꢀ function to abstract the 4′ꢀhydroxyl hydrogen from the
GalNAc (Fig. 5d). Substitution K86G in GalESp2 canꢀ sugar substrate. Based on sequence analysis, group 1
celled the ability of the enzyme to transform UDPꢀ epimerases, such as the E. coli GalE, have a corresponding
Glc/UDPꢀGal completely (Fig. 5a). This result indicates “Y300” residue. Group 3 epimerases, including WbgU
that Lys86 is vital to GalESp2 when functioning with nonꢀ from P. shigelloides [22] and WbpP from P. aeruginosa
acetylated substrates.
[23], have a corresponding G86 residue.
Our results clearly indicate that the determinants of
substrate specificity are not exclusively located directly on
the surface of the substrateꢀbinding pocket. The siteꢀ
directed mutagenesis data suggest that the Lys86 residue,
DISCUSSION
Sugars in capsular tetrasaccharide units of S. pneuꢀ which is not located in the GxxGxxG or YxxxK motif, is
moniae TIGR4 can be biosynthesized via the Leloir or necessary for GalESp2 to have its full activity. GalESp2
other pathways. Many genes can affect the biosynthesis of belonging to group 2 can transform to group 3 with the
capsular tetrasaccharide units, such as glucokinase amino acid substitution K86G. Since both GalESp1
(
GluK), phosphoglucomutase (Pgm), UTPꢀglucoseꢀ1P (group 1) and GalESp2 (group 2) from TIGR4 have relatꢀ
uridylyltransferase (GalU), galactokinase (GalK), UDPꢀ ed Lys86 residues, these results indicate that Lys86
galactose 4ꢀepimerase (GalE), etc. [24]. residues in GalESp1 (group 1) and GalESp2 (group 2) play
UDPꢀgalactose 4ꢀepimerase (GalE) is involved in a critical role in the activity of UDPꢀGlc/UDPꢀGal.
the biosynthesis of CPS of S. pneumoniae and plays an Substitution GalESp2 C300Y greatly decreased its activity
essential role in the virulence of some bacteria. A defiꢀ toward acetylated substrates, which suggests that GalESp2
ciency of the GalE enzyme is associated with many disꢀ belonging to group 2 can be transformed to group 1 with
eases, including an embryonicꢀlethal phenotype in fruit amino acid substitution C300Y. Compared to the amino
flies and type III galactosemia in humans. Considering acid sequence of other GalEs, the Lys86 is conserved in
the essential role of GalE in CPS synthesis and the S. group 2 but not in groups 1 and 3. Interestingly, when the
pneumoniae virulence, the characterization of this Gly102 (corresponding to Lys86 in GalESp2) in WbpP
enzyme may provide more information for controlling (GalE of P. aeruginosa) was changed to Lys, the WbpP
diseases caused by pneumococcal infection.
was transformed to group 2 from group 3 [23]. This illusꢀ
GalE from Pyrococcus horikoshii has been used to trates that having the amino acids Gly or Lys in these sites
develop a new process for enzymatic production of UDPꢀ may have decisive influences on the substrate specificity
Gal from UDP and is an inexpensive starting material of the enzymes.
[
25]. This work has shown the great potential of UDPꢀ
A single point mutation of a residue can change the
galactose 4ꢀepimerase in synthesis of compounds conꢀ activity of GalEs, such as L320C in A. nidulans, Y299C in
taining unnatural galactose derivatives, in drug target E. coli, and A209H in P. aeruginosa [28, 29]. This residue
design, and in vaccine development. Until now, GalE is thus called a gatekeeper, and there can be more than
from S. pneumoniae TIGR4 had not been studied.
one gatekeeper in a given GalE. The Lys86 residue could
The amino acid sequence alignment showed that be a new potential gatekeeper in GalESp2
both GalESp1 and GalESp2 from S. pneumococcus TIGR4
.
were more consistent with group 2 epimerases. Both were
predicted to epimerize acetylated and nonꢀacetylated Acknowledgments
substrates. However, our experimental results suggested
that only GalESp2 has both activities, while GalESp1 could
not epimerize the acetylated substrates UDPꢀGalNAc Program of Shandong Academy of Agricultural Sciences,
and UDPꢀGlcNAc. Innovation Program of agricultural technology in
This work was supported by Young Talents Training
Most studies of C4 epimerases have focused on refinꢀ Shandong Province 2015/16, National Natural Science
ing the catalytic mechanism involved, but few have examꢀ Foundation of China (No. 31770997, 31270983),
ined the molecular basis for their substrate specificity. Shandong Provincial Natural Science Foundation of
GalE variants from different organisms differ significantly China (No. ZR2016YL022), and SRF for ROCS and
in their DNA sequences yet are relatively homologous in SEM.
their quaternary structure and belong to the same enzyme
superfamily of shortꢀchain dehydrogenases (SDR) [26,
2
7]. The SDR enzyme family has two characteristic signaꢀ
ture sequences. One is GxxGxxG, which is contained
within an alternating α and β structure popularly known 1. Kadioglu, A., Weiser, J. N., Paton, J. C., and Andrew, P. W.
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