Fluorescent metal-organic frameworks based on mixed organic ligands: New candidates for highly sensitive detection of TNP

Article abstract of DOI:10.1039/c8dt03811b

In this study, by employing mixed organic ligands through rational design and metal ions as the initial reactants, a series of novel metal-organic frameworks (AHU-TW1 to 6) were synthesized by a solvothermal method. Herein, accessible Lewis base sites ori

Full text of DOI:10.1039/c8dt03811b

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Biochemical studies of a β-1,4-rhamnosly-
transferase from Streptococcus pneumonia
serotype 23F†
Cite this: DOI: 10.1039/c8ob02795a
Received 9th November 2018,
Accepted 7th January 2019
Hong Wang,‡a Siqiang Li,
‡
a,d Chenghe Xiong,a Guoxia Jin,c Zonggang Chen,a
a,b
Guofeng Gu *a and Zhongwu Guo
*
DOI: 10.1039/c8ob02795a
rsc.li/obc
A new β-rhamnoslytransferase Cps23FT from Streptococcus pneu- syltransferases (GTs). Thereafter, the repeating units are trans-
monia serotype 23F was expressed and characterized. Its enzy- located by flippase Wzx onto the cytoplasm membrane outer
matic activity and function were confirmed for the first time by uti- surface, where they polymerize under the catalysis of polymer-
lizing enzymatically prepared dTDP-Rha and chemically syn- ase Wzy to create CPS.3,7 In addition to CPSs, many exopolysac-
thesized Glcα-PP-(CH2)11-OPh as substrates. This reaction gave the charides and O-antigens are also biosynthesized through this
desired disaccharide Rhaβ-1,4-Glcα-PP-(CH2)11-OPh in a good iso- pathway.8
lated yield (67%), suggesting the potential of Cps23FT as a tool
To investigate and make use of the CPS biosynthesis
enzyme for the synthesis of complex oligosaccharides containing process, it is necessary to have access to the various GTs
difficult β-rhamnosyl linkages. Furthermore, site-directed muta- involved. Thus, bacterial GTs have attracted much attention in
genesis of Cps23FT disclosed that its 271DKD273 motif was critical recent decades and, as a result, many GTs have been identified
for the enzymatic activity and most likely the binding site for the and found wide applications.9,10 However, reports about
required divalent metal cation.
L-rhamnosyl-transferases (Rha-Ts) are currently rare to nearly
nonexistent.11–17 On the other hand, L-rhamnose (Rha) is an
extremely common sugar residue in bacterial polysaccharides,
whereas it is not observed in human glycans.18 For example,
most CPSs of various Streptococcus pneumoniae (pneumococ-
cus) serotypes, which are classified according to their CPS
structures, contain at least one Rha residue in their repeating
units.3 Therefore, the discovery and characterization of new
Rha-Ts are urgent and important for the investigation and
application of bacterial polysaccharides.
Furthermore, the β-linkage of L-Rha is one of the most
difficult glycosidic linkages to achieve in synthetic carbo-
hydrate chemistry, because both anomeric and steric effects
favour the formation of its α-glycosidic linkage.19
Consequently, Rha-Ts are not only useful for the study of CPSs
and their biosynthetic pathways but also valuable tools for the
chemoenzymatic synthesis of Rha-containing oligosaccharides
and/or polysaccharides9,10,20 and other rhamnosyl conjugates
that have found broader applications.21,22
To address the abovementioned problem, this research
aimed at discovering new bacterial Rha-Ts. In this regard, we
became interested in pneumococcus serotype 23F, as its CPS
repeating unit (1, Fig. 1) was composed of a repeating tetrasac-
charide unit containing D-glucose, D-galactose, L-rhamnose
(both α- and β-linkages), and phosphoglycerol.23 Bioinformatic
analysis of the genetic loci of serotype 23F CPS further
suggested that Cps23FT is the β-1,4-Rha-Ts that adds the first
Rha to Glcα-PP-Und,6,7 but this enzyme has not been expressed
Capsular polysaccharides (CPSs) are important bacterial viru-
lence factors that play a key role in bacterial survival, coloniza-
tion, evasion of the host immune system, etc.1,2 Due to their
important biological functions and highly conserved struc-
tures and presence in bacteria, CPSs have become useful
targets in the development of novel antibacterial strategies,
including vaccines. For instance, a variety of CPS vaccines or
CPS-based conjugate vaccines have been commercialized and
used in the clinic to prevent bacterial infections.3–5
The majority of these bacteria uses the Wzy-dependent
pathway for CPS biosynthesis.3,6,7 In this pathway, the assem-
bly of CPS repeating units is the first committed step realized
on the cytoplasm membrane inner surface by a series of glyco-
aNational Glycoengineering Research Center and Shandong Provincial Key
Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University,
72 Binhai Road, Qingdao 266237, China. E-mail: guofenggu@sdu.edu.cn
bDepartment of Chemistry, University of Florida, 214 Leigh Hall, Gainesville,
Florida 32611, USA. E-mail: zguo@chem.ufl.edu
cCollege of Chemistry, Chemical Engineering and Materials Science,
Shandong Normal University, 88 Wenhua Dong Lu, Jinan 250014, China
dSchool of Biological and Food Engineering, Huanghuai University,
76 Kaiyuan Road, Zhumadian 463000, China
†Electronic supplementary information (ESI) available: Experimental pro-
cedures, primers used for gene cloning, protein analysis results, enzyme reaction
condition optimization and kinetics results, product MS and NMR spectra. See
DOI: 10.1039/c8ob02795a
‡These authors contributed equally to this article.
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conjugate biosynthesis.27 Inspired by these discoveries, we
designed and chemically synthesized
a
glycolipid
3
(Glcα-PP-C11-OPh, Scheme 1), which had
a
11-phenoxy-
undecan-1-yl lipid as an Und surrogate α-linked to the anome-
ric position of Glc through a diphosphate bridge, to serve as a
potential substrate and Rha acceptor for Cps23FT. If it works,
glycolipid 3 can have multiple advantages over Und-PP-Glc 2.
First, the chemical synthesis of 3 (Scheme S1 and Fig. S6–10,
ESI†) is simple as compared to that of 2. Second, 3 should be
much more water-soluble than 2 to facilitate homogeneous
reactions in aqueous media. Third, the shortened lipid chain
and the presence of an UV-active phenyl group in the lipid of 3
would significantly simplify the analysis and purification of
reaction products. Finally, the shorter lipid and improved
water solubility of 4, as compared to its Und counterpart,
would make NMR and MS studies of the product much more
straightforward.
Fig. 1 The structure of the pneumococcus serotype 23F CPS.
and functionally verified yet. Moreover, gene sequence analysis
of Cps23FT revealed that it does not contain transmembrane
domains, making it a water-soluble enzyme particularly suit-
able for chemoenzymatic synthesis of related oligosaccharides.
Consequently, this work was focused on Cps23FT.
A recombinant plasmid pET-22b-cps23FT encoding the full-
length Cps23FT and a C-terminal His6 tag was constructed by
the standard protocols. Then, the Cps23FT-His6 fusion protein
was expressed using Escherichia coli BL21 and purified to hom-
ogeneity, as proved by SDS-PAGE, in two steps using a Ni-
column and a Q-column, respectively. The SDS-PAGE and
western blot (Fig. S1A and B, ESI†) results also showed that the
protein had an apparent molecular weight of 46 kDa, which
was consistent with the calculated value (46 193 Da).
Preliminary studies on the enzymatic activity of Cps23FT to
catalyze β-rhamnosylation of 3 were carried out in the 4-mor-
pholineethanesulfonic acid hydrate (MES) buffer (50 mM, pH
7.5) containing 10 mM MnCl2, 0.5 mM dTDP-Rha, and 1 mM
3, and the concentration of purified Cps23FT was about 400 μg
mL−1. The reactions were kept at 16 °C for 1 h and then sub-
jected to LC-ESI-HRMS analysis. The observed prominent peak
with an m/z ratio of 731.2412 [M − H]1− (Fig. S12, ESI†) was
consistent with the calculated exact mass (731.2450) of the
expected 4, suggesting the enzymatic activity of Cps23FT to cat-
alyze the reaction shown in Scheme 1.
In the literature, there have been very few reports about
functional characterization of Rha-Ts. One of the major issues
was related to difficult access to Rha donors, such as thymi-
dine diphosphate-L-rhamnose (dTDP-Rha), which are not com-
mercially available, whilst their chemical synthesis is not
viable either. To our advantage, we have recently established a
one-pot-four-enzyme strategy for the efficient and scalable syn-
thesis of dTDP-Rha,24 which would enable the study and
characterization of Cps23FT. Another barrier that has been
hindering the investigation of Rha-Ts is the difficulty to access
sugar acceptors recognized by Rha-Ts. The glycosyl acceptors
for GTs involved in CPS biosynthesis are usually derivatives of
The product 4 was further characterized with 1D 1H, 13C
and 31P and 2D 1H–1H COSY, HSQC and HMBC NMR data
(Fig. S13–19, ESI†). For example, the newly generated β-Rha-
1
1,4-Glc linkage in 4 was confirmed with its H-coupled gHSQC
1
and gHMBC spectra (Fig. S17 and S18, ESI†). The JC-1,H-1
coupling constant (162 Hz) of the Rha residue in the 1H-
coupled gHSQC of
4
indicated unambiguously its
β-configuration.28 A distinct correlation signal between H-1Rha
and C-4Glc in its gHMBC spectrum confirmed the correct Rha-
1,4-Glc linkage in 4.
undecaprenyl diphosphate (Und-PP),25 which contain
a
55-carbon lipid that anchors into the cytoplasmic mem-
brane.26 For example, the natural Rha acceptor for Cps23FT is
Und-PP-Glc (2, Fig. 2). Und-P is difficult to obtain and is thus
notoriously expensive; in addition, it has extremely poor solu-
bility in aqueous media. This has created a number of issues
for the assay of these GTs, such as difficulty in the preparation
of homogenous solutions for enzymatic reactions and final
product purification and characterization.
As outlined in Scheme 1, the reaction of 3 with dTDP-Rha
catalyzed by Cps23FT gave thymidine diphosphate (dTDP) as a
byproduct. Owing to its stronger ability of UV absorption than
that of 4, dTDP was selected to monitor the reaction and
However, previous studies have demonstrated that sugar
derivatives with shorter lipid chains can sometimes be
accepted by GTs involved in CPS and other bacterial glyco-
Scheme 1 The designed Rha acceptor 3 and its rhamnosylation used
Fig. 2 The structure of the natural Rha acceptor Und-PP-Glc 2.
to characterize and analyze the activity of Cps23F.
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analyse the enzyme kinetics (Fig. S20, ESI†). To examine the
influences of pH, temperature and metal ion, the reaction of 3
with dTDP-Rha in the presence of Cps23FT was carried out at
varied pH, temperature, and metal ion concentrations, with
heat-deactivated Cps23FT as the control. As shown in Fig. 3A,
Cps23FT exhibited the best catalytic activity at pH ∼7.5 and
stronger basic or acidic conditions caused a significant
decrease in the activity. However, Cps23FT could tolerate a
relatively wide range of temperatures, as >80% of its activity
was kept at 16–37 °C with the maximal activity observed at
16 °C (Fig. 3B). The results shown in Fig. 3C suggested that
Cps23FT required a divalent metal cation, such as Mn2+, Mg2+
and Ca2+ (5 mM), for its catalytic activity. In the presence of
Ag+, Cu2+, Fe2+, Ni2+ and Zn2+ or K+ and Na+ (5 mM), Cps23FT
showed none to only 40% activity. These results indicated that
Cps23FT might belong to the GT-A family. Because under the
Fig. 4 Results of enzyme kinetics studies of Cps23FT for 3 (A) and for
dTDP-Rha (B). Each error bar represents the standard deviation of three
experiments.
same conditions Mn2+ was the best cation to activate Cps23FT, Vmax values were calculated from Michaelis–Menten plots as
the influences of Mn2+ concentration on the reaction were shown in Fig. 4. The Km values of 3 and dTDP-Rha were
then examined in more detail. It was observed that Cps23FT 770.00 77.06 and 290.30 17.17 μM, and their Vmax values
showed the maximal activity with 8 mM Mn2+ (Fig. 3D). were 23.55 0.74 and 30.41 0.59 μM min−1, respectively. The
Therefore, the optimal conditions for Cps23FT to catalyze the lower Km value of Cps23FT for dTDP-Rha, as compared to the
Rha transfer reaction were established as 8 mM Mn2+ at pH 7.5 Km values of Rha-Ts from other bacteria, such as
and 16 °C.
Saccharopolyspora spinose SpnG (600.00 μM),12 indicated that
The above-optimized reaction conditions were utilized to Cps23FT had a higher affinity to dTDP-Rha than other
analyze the enzyme kinetics of Cps23FT, with the initial vel- reported similar enzymes.
ocities of the reaction obtained with varied Cps23FT concen-
trations (25, 50, 100, 200 and 400 μg mL−1) and reaction times thesis employing the optimized reaction conditions. The
(5–80 min). Finally, reaction conditions using 50 μg mL−1 of desired disaccharide product
was obtained from the
In addition, we also performed a large-scale enzymatic syn-
4
Cps23FT and 10 min of incubation time were selected for the Cps23FT-catalyzed reaction of 3 with dTDP-Rha in multi-milli-
study. To measure the Km and Vmax values for 3 and gram quantity and a 67% isolated yield after HPLC purifi-
dTDP-Rha, the enzymatic reactions were examined using satu- cation. The product purity was >95% (Fig. S11, ESI†).
rated dTDP-Rha (0.5 mM) and 0.1–6.4 mM 3 or saturated 3 Nonetheless, we believe that the reaction can be achieved in
(2 mM) and 0.12–2.0 mM dTDP-Rha, respectively. The Km and even larger scales and better yields upon additional optimiz-
ation of the workup and purification protocols.
GTs have two predominant families, GT-A and GT-B, and
many bacterial GTs belong to GT-A.29 A common feature of the
GT-A family is the presence of a short and conserved DXD
motif, which is required for binding with a divalent metal
cation.30 The divalent cation-dependence of Cps23FT indicated
that it might belong to the GT-A family. If this is true, Cps23FT
should contain a DXD motif or its variant. Upon analyzing the
sequence of Cps23FT (Fig. S21, ESI†), we found two potential
DXD motifs, i.e., 97DED99 and 271DKD273. Although crystal
structures of the catalytic domains of some GTs had been
reported, they did not come up with a consensus location of
the divalent metal cation-binding motif. To identify which one
of the two DXD motifs in Cps23FT is involved in its catalytic
activity, four Cps23FT mutants, D97A, D99A, D271A and
D273A (Table S1, ESI†), were expressed and then subjected to
in vitro activity assays according to above-established protocols.
It was expected that substituting the aspartic acid residue with
an alanine at the catalytic domain of Cps23FT would decrease
its divalent metal cation binding ability and thus its catalytic
activity. Among the four mutants, D97A and D99A remained
Fig. 3 The influences of pH (A), temperature (B), ion (C), and Mn2+ con-
centration (D) on the relative enzymatic activities of Cps23FT to catalyze
almost as active as the wild-type enzyme, while D271A and
D273A showed significantly decreased activity (Fig. 5). These
the reaction of dTDP-Rha with 3. Each error bar represents the standard
deviation of three experiments.
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the Science and Technology Development Project of Shandong
Province (No. 2016GGH4502).
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Fig. 5 Relative enzymatic activities of Cps23FT and its D97A, D99A,
D271A, and D273A mutants. The reactions were carried out in 50 mM
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In summary, with the help of newly established methods
for efficient chemical and chemoenzymatic syntheses of
dTDP-Rha as a Rha donor and glycolipid 3 carrying a truncated
lipid as a Rha acceptor for Rha-Ts, we identified a novel
β-1,4-Rha-Ts, Cps23FT derived from pneumococcus serotype
23F and performed detailed biochemical studies on it.
Moreover, Cps23FT, was proved to be a potentially useful tool
enzyme for constructing the difficult β-Rha linkage, especially
for the synthesis of oligosaccharides containing the Rhaβ1,4-
Glc motif. In the meantime, we have also demonstrated that
Cps23FT could accept 3, which carried an artificial lipid, as a
Rha acceptor, suggesting that Cps23FT can tolerate structural
modification of the Rha acceptor. Thus, this work represents
the first biochemical characterization of Cps23FT and has
presented the first direct and unambiguous evidence for its
bioactivity to catalyze β-rhamnosylation and its function in the
process of pneumococcal CPS biosynthesis.
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cation was necessary for the catalytic activity of Cps23FT, indi-
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has laid the foundation for gaining more insight into the func-
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