One-pot four-enzyme synthesis of thymidinediphosphate-l-rhamnose

Article abstract of DOI:10.1039/c6cc08366h

A new, robust one-pot four-enzyme synthetic method was developed for thymidinediphosphate-l-rhamnose starting from d-glucose-1-phosphate. The enzymes, Glc-1-P thymidylyltransferase, dTDP-Glc-4,6-dehydratase, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase and dTDP-4-keto-Rha reductase were derived from Streptococcus pneumonia serotype 23F, expressed in Escherichia coli, and studied in detail to provide the first direct evidence for their functions.

Full text of DOI:10.1039/c6cc08366h

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One-Pot Four-Enzyme Synthesis of Thymidinediphosphate-L-
rhamnose
Siqiang Li,a Hong Wang,a Juncai Ma,a Guofeng Gu,a,* Zonggang Chena,* and Zhongwu Guo b,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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Abstract. A new, robust one-pot four-enzyme synthetic method was of dTDP-Rha utilizing dTDP-D-glucose (dTDP-Glc), a biosynthetic
developed for thymidinediphosphate-L-rhamnose starting from D- intermediate of dTDP-Rha, and crude enzymes.29-32 However,
glucose-1-phosphate. The enzymes, Glc-1-P thymidylyltransferase, this method is not suitable for large scale synthesis since dTDP-
dTDP-Glc-4,6-dehydratase, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase Glc is expensive and difficult to obtain. A less costly synthesis
and dTDP-4-keto-Rha reductase were derived from Streptococcus for dTDP-Rha was developed by the use of six crude enzymes.33
pneumonia serotype 23F, expressed in Escherichia coli, and studied However, the quality of enzymes was uncontrollable, and the
in detail to provide the first direct evidence for their functions.
product purification was challenging. James and coworkers34,35
described a two-step four-enzyme synthesis, but the reactions
were poorly characterized. For example, the reaction yield and
the product purity were not described in the paper. Moreover,
this synthesis needed two purification steps, which is still
demanding. Clearly, a more robust synthetic method for dTDP-
Rha using well-characterized enzymes and low-cost and easily
accessible starting materials is highly desirable.
We report herewith a new, simple, and effective one-pot four-
enzyme synthesis of dTDP-Rha from commercial D-glucose-1-
phosphate (Glc-1-P) and thymidine triphosphate (dTTP). The
enzymes used included Glc-1-P thymidylyltransferase Cps23FL,
dTDP-Glc-4,6-dehydratase Cps23FN, dTDP-4-keto-6-deoxy-Glc-
3,5-epimerase Cps23FM, and dTDP-4-keto-Rha reductase
Cps23FO derived from Streptococcus pneumonia serotype 23F.
Although the functions of Cps23FL, Cps23FN, Cps23FM and
Cps23FO were proposed,36 no direct biochemical evidence for
their activities or detailed studies and synthetic applications of
the pure enzymes have been reported previously.
L-Rhamnose (Rha) is a common component of many bacterial
polysaccharides, e.g., lipopolysaccharides1,2 and extracellular,3
capsular4 and cell wall polysaccharides,5,6 which play a pivotal
role in various biological events. To study these polysaccharides
and related glycoconjugates, it is necessary to have access to
the molecules in structurally homogeneous and defined forms,
which is difficult to achieve via isolation of natural products.
Thus, chemical and enzymatic synthesis of polysaccharides and
their analogues has been a hot topic. In this regard, enzymatic
synthesis is especially attractive due to the complex structure
of the synthetic targets and the difficulty to create some
glycosidic bonds, such as that of β-linked Rha, via chemical
glycosylation. For enzymatic synthesis of Rha-containing glyco-
conjugates, thymidine diphosphate-L-rhamnose (dTDP-Rha) is a
key substrate,7-13 used by rhamnosyltransferases (Rha-Ts) as the
rhamnosyl donor to be attached to sugar acceptors.14
However, dTDP-Rha is not easily accessible, and its chemical
synthesis remains a significant challenge.13 As a result, much
recent research effort has been put on its enzymatic synthesis.
dTDP-Rha biosynthesis is conserved in bacteria,15 involving
enzymes such as RmlA, RmlB, RmlC, and RmlD.16-26 Marumo,13
Graninger27 and Steiner28 labs reported an enzymatic synthesis
Expression and purification of the enzymes involved in dTDP-
Rha biosynthesis in S. pneumonia serotype 23F. Capsular
polysaccharides derived from various S. pneumonia serotypes
contain Rha,37 and their biosynthesis uses dTDP-Rha as the Rha
donor.24,38 In turn, the biosynthesis of dTDP-Rha starting from
Glc-1-P (Scheme 1)7,13,28 involves four enzymes, including Glc-1-P
thymidylyltransferase, dTDP-Glc-4,6-dehydratase, dTDP-4-keto-
6-deoxy-Glc-3,5-epimerase, and dTDP-4-keto-Rha reductase.
In S. pneumonia type 23F, proteins Cps23FL, Cps23FN, Cps23FM,
and Cps23FO were proposed to correspond to above enzymes,
respectively.36 To study these gene products in detail and their
applications to dTDP-Rha synthesis in vitro, we cloned cps23FL,
cps23FM, cps23FN, and cps23FO genes from S. pneumonia 23F
by the conventional protocols, using primers listed in Table S1,
a. National Glycoengineering Research Center and Shandong Provincial Key
Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University,
27 Shanda Nan Lu, Jinan 250100, China.
b. Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville,
Florida 32611, United States.
* Corresponding author emails: zguo@chem.ufl.edu; guofenggu@sdu.edu.cn;
chenzg@sdu.edu.cn.
Electronic Supplementary Information (ESI) available: Experimental procedures,
primers used for gene cloning, protein analysis results, enzyme reaction condition
optimization and kinetics results, product MS and NMR spectra. See
DOI: 10.1039/x0xx00000x
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and used them to express corresponding proteins in Escherichia
coli. In addition, a His-6 tag was introduced to the N-terminus
of these proteins to facilitate their purification. SDS-PAGE and
western blot studies (SI Figures S1A and S1B) of the purified
proteins showed that their molecular weights (34.3, 41.1, 24.4
and 34.5 KD, respectively) agreed with the predicted molecular
weights of Cps23FL, Cps23FN, Cps23FM and Cps23FO. Figure S1
also revealed that the proteins were essentially homogeneous.
The purified proteins were also analysed with the BCA protein
quantification kit to determine the protein expression levels of
Cps23FL, Cps23FN, Cps23FM, and Cps23FO, which were 107,
91, 65, and 94 mg/litter, respectively. The pure proteins were
stable in Tris-HCl buffer containing 30% glycerol at pH 7.5 and
showed no activity loss after storing at -20 oC for 2 months.
View Article Online
DOI: 10.1039/C6CC08366H
(A)
(B)
Figure 1. Results of indirect assays of the enzymatic activities of
Cps23FL, Cps23FN, Cps23FM, and Cps23FO. (A) Reaction between
Glc-1-P and dTTP in the presence of Cps23FL or Cps23FN alone or
Cps23FL plus Cps23FN. (B) Conversion of product obtained with
Cps23FL and Cps23FN in the presence of Cps23FM or Cps23FO
alone or both Cps23FM and Cps23FO. Each data point represents
the mean ± standard error of three experiments
To optimize the reaction conditions for Cps23FL, it was treated
with Glc-1-P and dTTP at varied pH, temperature, and metal ion
concentration, with heat-deactivated Cps23FL as a control. The
reaction was monitored by the malachite green colorimetric
assay41 detecting the generated pyrophosphate (PPi) upon its
degradation by an inorganic pyrophosphatase YIPP to generate
phosphate. To avoid potential influences of YIPP concentration
on the assays, YIPP was utilized in large excess. As depicted in
Figures S4A and S4B, Cps23FL had the maximal catalytic activity
o
at pH 7.5 and 25 C. However, Cps23FL tolerated a relatively
wide range of temperature, as >80% of its activity retained from
o
16 to 40 C. It was also found that divalent metal cations were
required for its catalytic activity and the optimal cation was
Mg2+ (Figure S4C). Moreover, the activity of Cps23FL reached
its maximum at 0.63 to 40 mM of Mg2+ (Figure S4D). Thus, for
future study and application of Cps23FL, its reaction conditions
were set at pH 7.5, 37 oC, and 5.0 mM of Mg2+.
To evaluate the reaction kinetics of Cps23FL, its Km and Vmax
values for both substrates were determined. Initial velocities of
the reaction under optimized conditions were obtained using
Scheme 1. Biosynthetic pathway of dTDP-Rha in S. pneumonia
Biochemical studies of Cps23FL, Cps23FM, Cps23FN, and
Cps23FO. Initially, we briefly evaluated the activities of the
obtained proteins by indirect assays. It was found that after
adding Cps23FL and Cps23FN, the mixture of Glc-1-P, dTTP,
and NAD+ showed a significantly increased absorbance at the
wavelength of 320 nm than the control groups containing no or
only one enzyme (Figure 1A), indicating the formation of 4-keto
sugar from the first two reactions depicted in Scheme 1.
Furthermore, upon treatment with Cps23FM and Cps23FO, the
mixture derived from the above reactions showed a decreased
absorbance at 340 nm than the controls (Figure 1B), indicating
the consumption of NADH by the last reaction in Scheme 1.
These results suggested that Cps23FL, Cps23FN, Cps23FM, and
varied dTTP concentrations (0.01
Glc-1-P (1 mM) or varied Glc-1-P concentrations (0.05
～
0.08 mM) and saturated
0.40
～
mM) and saturated dTTP (1 mM). The data were employed to
create Michaelis-Menten plots and derive Km and Vmax, which
were found to be 9.57 ± 1.58 μM and 4.15 ± 0.16 μM/min for
dTTP, and 18.04 ± 1.48 μM and 8.36 ± 0.01 μM/min for D-Glc-
1-P, respectively. Comparing the Km values of Cps23FL to that
of RmlA derived from other bacteria, such as Mycobacterium
tuberculosis,42 Salmonella enterica LT243 and Aneurinibacillus
thermoaerophilus27 (0.02, 0.02, 0.03 mM for dTTP; 0.07, 0.11,
0.20 mM for D-Glc-1-P, respectively), the Km value of Cps23FL
for D-Glc-1-P was significantly lower, indicating that Cps23FL
had higher affinity to D-Glc-1-P. However, there was not a
drastic difference among the Km values for dTTP.
To verify the enzymatic activity of Cps23FN, it was reacted with
a mixture of Cps23FL, Glc-1-P, dTTP and NAD+ under conditions
described above. The product was obtained in a 71% yield
after purification by HPLC and by a G10 column. Its HR MS
(Figure S5) gave an ion peak at m/z 545.0546, which agreed
well with the molecular weight of dT4k6dG (C16H22N2O15P2 [M-
H]-, 545.0574). Its 1H and 13C NMR spectra (Figure S6) were also
identical to that of the reported dT4k6dG.44-46 Both HR MS and
Cps23FO might possess the desired enzymatic activities
.
Next, each enzyme was examined more closely. To confirm the
activity of Cps23FL, the product of its reaction with Glc-1-P and
dTTP was isolated by Q anion exchange chromatography. The
ion peak at m/z 563.0705 of its high resolution mass spectrum
(HRMS, Figure S2) agreed with the molecular weight of dTDP-
Glc (563.0679 for C16H25N2O16P2 [M-H]-1), and its 1H and 13C NMR
spectra (Figure S3) were the same as that in the literature.39,40
Therefore, both MS and NMR data confirmed the product as
dTDP-Glc. The reaction yield was about 86% based on Glc-1-P.
This study proved unambiguously the activity of Cps23FL as an
enzyme to catalyze the conversion of Glc-1-P to dTDP-Glc.
2 | J. Name., 2012, 00, 1-3
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NMR data proved the production of dT4k6dG and thus the the reaction equilibrium towards the final prodVuiecwt AlratictleerOnolinne.
DOI: 10.1039/C6CC08366H
Thereafter, Cps23FL was removed by ultrafiltration, followed
dTDP-Glc-4,6-dehydratase activity of Cps23FN.
Subsequently, the catalytic property of Cps23FN was studied in by adding Cps23FN, Cps23FM, and Cps23FO (2 μM, 82.2, 48.8
detail by using pure dTDP-Glc as substrate. The reaction was and 69.0 mg/L, respectively). The reaction was monitored with
examined by spectrophotometric analysis of resulting dT4k6dG, HPLC to reveal the generation of dTDP-Rha in a good yield
which had specific absorption at 320 nm under basic condition, (Figure 2). After completion, the reaction was quenched and
caused by enediolate formed from the deprotonation of keto the enzymes were removed by ultrafiltration. The final product
sugar.27,33,47-49 In these experiments, mixtures of dTDP-Glc and was purified by HPLC, desalted on a G10 column, and then
NAD+ were incubated with Cps23FN or the heat-deactivated lyophilized to produce dTDP-Rha as a white solid. The overall
enzyme (negative control), respectively, and then quenched isolated yield was 47% based on Glc-1-P. However, dTDP-Rha
with 1 M NaOH. We found that Cps23FN showed the highest was not easily separated from side products such as dTMP and
o
catalytic activity at 37 C and pH 7.5 (Figure S7), while various NADH by Q anion exchange chromatography. The reaction yield
cations had a negligible impact on the reaction. Moreover, the was further improved to 63% when the operation to remove
initial velocities of Cps23FN-catalyzed reactions under optimal Cps23FL by ultrafiltration was eliminated, probably as a result
conditions were measured with different concentrations (0.1 of decreased intermediate loss. It is also worth noting that
～1.0 mM) of dTDP-Glc. The results were used to obtain the Km using excessive dTTP (ca. 1.5 equiv) in the reaction was
(0.86 ± 0.11 mM) and Vmax (0.07 ± 0.004 mM/min) of Cps23FN. beneficial, because it not only improved the reaction yield but
The higher Km value of Cps23FN than that of RmlB from other also simplified product purification by exhausting Glc-1-P that
bacteria, such as S. enterica LT2 (0.427 mM),50 M. tuberculosis was difficult to separate from dTDP-Rha.
(0.050 mM)48 and E. coli RmlB (0.070 mM),51 indicated that
Cps23FN had lower affinity to dTDP-D-glucose.
Detailed study of Cps23FM or Cps23FO individually turned out
dTDP-Rha
to be difficult since (1) Cps23FM alone without Cps23FO had a
low rate of substrate conversion (ca. 3%) and (2) its product,
TDP-4-keto-L-Rha, was not stable enough to be isolated.52 Thus,
the two enzymes were studied in conjugation. To confirm the
enzymatic activities of Cps23FM and Cps23FO disclosed above,
they were incubated with pure dT4k6dG and NAD+/NADH. The
reaction mixture was subjected to HPLC analysis, revealing a
new product that was collected and characterized. Its HR MS
showed a peak at m/z 547.0711 (Figure S8) that agreed with
the calculated molecular weight of dTDP-Rha (547.0736 for
*
Figure 2. HPLC diagrams of (A) NAD+, (B) NADH, (C) dTTP, (D)
dTDP-Glc, (E) dT4k6dG, (F) dTDP-Rha, (G) reaction mixture of
heat-deactivated enzymes, and (H) reaction mixture for one-
1
C16H25N2O15P2 [M-H]-). Its H, 13C, and 31P NMR spectra (Figure
pot four-enzyme synthesis of dTDP-Rha from Glc-1-P. *dTMP
S9) were in accordance with the structure of dTDP-Rha.13 The
production of dTDP-Rha from this reaction proved ultimately
the dTDP-4-keto-6-deoxy-Glc-3,5-epimerase and dTDP-4-keto-L-
Rha reductase activities of Cps23FM and Cps23FO.
This study has established a straightforward, efficient, and
economical one-pot synthetic method for dTDP-Rha starting
from inexpensive and commercially available Glc-1-P, by using
enzymes cloned from S. pneumonia serotype 23F. Compared
to the preparation methods reported in the literature, this
strategy has several advantages. For example, in addition to
the inexpensive starting material and abolition of intermediate
isolations through one-pot synthesis, the reaction progress
could be easily monitored and examined, and the final product
purification was simple, due to the use of pure enzymes in the
synthesis. Therefore, this synthetic method has the potential
of being used for large scale synthesis. For instance, dTDP-Rha
was prepared in hundred milligram scales in the authors’ lab.
Moreover, the enzymes involved, including Cps23FL, Cps23FN,
Cps23FM and Cps23FO, were purified and examined in detail,
which provided the first direct biochemical evidence for the
functions of these gene products and formed the foundation
for more in-depth studies. Overall, easy access to dTDP-Rha
would facilitate Rha-T-based chemoenzymatic synthesis of Rha-
containing oligosaccharides, polysaccharides, rhamnolipids,54,55
and related conjugates which should be very useful for various
biological studies and applications.
One-pot four-enzyme synthesis of dTDP-Rha from Glc-1-P.
After the properties of Cps23FL, Cps23FN, Cps23FM, and
Cps23FO were evaluated in detail and their enzymatic activities
were confirmed, we explored their application to the synthesis
of dTDP-Rha. As the optimized reaction conditions for these
enzymes, such as pH (7.5) and temperature (37 oC), are similar,
we were interested in a one-pot multiple enzyme synthetic
protocol. This would have several advantages, e.g., reducing
the number of purification steps and avoiding the isolation of
relatively unstable intermediates.
Our initial synthetic design was to simultaneously mix all of the
substrates, reagents, and enzymes in one pot, but it gave only
a very low yield (~1%) of dTDP-Rha, probably because of the
inhibition of dTDP-Rha formed from the reaction on Cps23FL.53
We found that the reaction yields was significantly improved
when the enzymes were added in two portions. First, a
solution of Glc-1-P (3 mM), dTTP (5 mM), NAD+ (0.1 mM), and
NADH (6 mM) in a HEPES buffer (30 mM, pH 7.5) containing 5
mM of MgCl2 was incubated with Cps23FL (2 μM, 68.6 mg/L) at
o
37 C for 15 minutes. Excessive NADH was used here to drive
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28. K. Steiner, R. Novotny, D. B. Werz, K. Zarschler, P. H. Seeberger,
Acknowledgement.
This work was supported by grants from the National Natural
Science Foundation of China (21672129 and 21472114).
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