Phosphomannose isomerase/GDP-mannose pyrophosphorylase from Pyrococcus furiosus: A thermostable biocatalyst for the synthesis of guanidinediphosphate- activated and mannose-containing sugar nucleotides

Article abstract of DOI:10.1039/b822794b

Herein we present an analysis of the chemical function of a recombinant bifunctional phosphomannose isomerase/GDP-mannose pyrophosphorylase (manC) from Pyrococcus furiosus DSM 3638 and its use in the synthesis of guanidinediphospho-hexoses and a range of

Full text of DOI:10.1039/b822794b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Phosphomannose isomerase/GDP-mannose pyrophosphorylase from
Pyrococcus furiosus: a thermostable biocatalyst for the synthesis of
guanidinediphosphate-activated and mannose-containing sugar nucleotides†
Rahman M. Mizanur and Nicola L. B. Pohl*
Received 19th December 2008, Accepted 4th March 2009
First published as an Advance Article on the web 27th March 2009
DOI: 10.1039/b822794b
Herein we present an analysis of the chemical function of a recombinant bifunctional phosphomannose
isomerase/GDP-mannose pyrophosphorylase (manC) from Pyrococcus furiosus DSM 3638 and its use
in the synthesis of guanidinediphospho-hexoses and a range of nucleotidediphospho-mannoses. This
enzyme is unusually promiscuous in both its nucleotide triphosphate (NTP) and sugar-1-phosphate
acceptance. It accepts all five naturally occurring NTPs (ATP, CTP, GTP, dTTP and UTP) and a range
of sugar-1-phosphates (glucose-, mannose-, galactose-, glucosamine-, N-acetylglucosamine- and
fucose-1-phosphate). A truncated GDP-mannose pyrophosphorylase domain of the whole length
enzyme showed almost 100-fold less sugar nucleotidyltransferase activity with only GTP and mannose
1-phosphate as substrates. The temperature stability and inherently broad substrate tolerance of this
archaeal enzyme make it an effective reagent for the rapid chemoenzymatic synthesis of a range of
natural and unnatural sugar nucleotides that are challenging to make by chemical means alone.
mannoses, although rarely found in nature, have also been used as
Introduction
substrates for structural studies of glycosyltransferases.8
Most monosaccharides are activated as sugar nucleotides before
Several mesophilic bacterial and eukaryotic GDP-mannose
pyrophosphorylases have been reported.6,9 A recombinant version
of the bacterial enzyme from Salmonella enterica has been
shown to be effective in the enzymatic synthesis of natural
GDP-mannose9j–l as well as azido-substituted versions of the
sugar nucleotide.9m One crenarchaeal GDP-mannose synthesizing
gene from the hyperthermophilic Sulfolobus solfataricus has been
identified, but the resulting protein produced only GDP-mannose
and not GDP-glucose.10 This inflexible crenarchaeal protein is
actually more closely related in sequence to the corresponding
protein from Arabidopsis thaliana than to that of the euryarchaea
Pyrococcus.
Only a few of the type II phosphomannose isomerases (PMIs),
bifunctional prokaryotic proteins with both PMI (E.C. 5.3.1.8)
and GMP (E.C.2.7.7.22) activities, however, have been reported.
Enzymes from several bacterial species that include Burkholde-
ria cenocepacia BceAJ9h, Pseudomonas aeruginosa AlgA9g, Xan-
thomans campestris XanB,9i Gluconobacter xylinum AceF,9f and
Helicobacter pylori HP00439b have been studied so far. The PMI
motif of the bifunctional PMI-GMP also has been proposed as
a target for the development of inhibitors.9k The PMI motif of
PMI-GMP catalyses the reversible conversion of D-fructose-6-
phosphate into D-mannose-6-phosphate, which is then isomerized
to mannose-1-phosphate (Man1P) by a phosphomannomutase;
the GMP motif then converts the Man1P into GDP-mannose in
the presence of GTP and a divalent cation (Scheme 1).
their incorporation into glycoconjugates and cell wall polysac-
charides by glycosyltransferases. Many of these glycosyltrans-
ferases employ uridine- or deoxythymidine-diphosphosugars, and
therefore most biosynthetic efforts have focused on accessing
these two classes of sugar nucleotide.1 However, there are many
mannose-transferring enzymes that form cell wall components
such as lipoarabinomannan and other key oligosaccharides uti-
lizing guanidinediphosphate (GDP)-activated sugar nucleotides.2
The enzymes that make these GDP-mannose building blocks are
necessary for the survival of eukaryotes.3 Although not vital for
pathogens such as Leishmania, these enzymes are required for their
virulence and are therefore a proposed target for therapeutics.4
Detailed studies of these pathways will require both activated man-
noses and analogous GDP-sugars. For example, recently chemical
synthesis has afforded fluorescent and azido-substituted GDP-
mannose analogs for probing mannosyltransferase activities.5,9m
A key enzyme for the synthesis of GDP-mannose is GDP-
mannose pyrophosphorylase (GMP), which catalyzes the synthe-
sis of GDP-mannose from mannose-1-phosphate and GTP with
the release of pyrophosphate (Scheme 1). The activated mannose
is the precursor for mannosylation of N-linked glycoproteins and
glycosylphosphoinositol anchors6 and is an essential metabolic
intermediate for the biosynthesis of other GDP-sugars such
as GDP-fucose, GDP-colitose, GDP-talose, GDP-perosamine
and GDP-D-rhamnose.7 In addition to GDP-mannose, NDP-
In contrast to bacterial enzymes, surprisingly no functional
PMI-GMP activity has been reported from archaea, the third
branch of life. A few bacterial and archaeal nucleotidyl trans-
ferases have been shown to be highly flexible with regard to their
nucleoside triphosphate substrates.9m,11 For instance, ST0452 of
the Sulfolobus tokodaii accepts all four major deoxyribonucleoside
Department of Chemistry and Plant Sciences Institute, Gilman Hall, Iowa
State University, Ames, Iowa 50011-3111. E-mail: npohl@iastate.edu
† Electronic supplementary information (ESI) available: Detailed experi-
mental procedures and extra figures. See DOI: 10.1039/b822794b
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Scheme 1 Synthesis of GDP-mannose from fructose-6-phosphate by a bifunctional phosphomannose isomerase/GDP-mannose pyrophosphorylase
(manC) and phosphomannomutase (PMM).
triphosphates (dTTP, dATP, dCTP and dGTP) and UTP as the
only ribonucleoside triphosphate in the presence of glucose-1-
phosphate (Glc1P). In addition to Glc1P, ST0452 also accepts
N-acetylglucosamine-1-phosphate (GlcNAc1P) in the presence of
UTP and dTTP.11a Substrate flexibility studies of three bacterial
thymidyltransferases—Cps2L from Streptococcus pneumoniae R6,
RmlA from S. mutans and RmlA3 from Aneurinibacillus ther-
moaerophilus DSM 10155—show them to accept NTPs (ATP,
CTP, GTP, dTTP and UTP) in the presence of G1c1P to thus
produce NDP-glucoses.11b An engineered version of Cps2L from
S. pneumoniae, designated as the Q24S mutant, was shown to
be active in the synthesis of various NDP-sugars including NDP-
mannoses except CDP-mannose.11c The GMP (manC) from E. coli
was reported to activate M1P to form GDP-, ADP-, CDP- and
UDP-mannose to varying degrees but does not accept dTTP. In
addition, this enzyme can synthesize GDP-glucose and GDP-2-
deoxyglucose.9a However, in addition to their thermo-sensitivity,
none of the enzymes were reported to be highly effective in the
synthesis of a range of GDP-sugars and NDP-mannoses.
The thermal vent archaea P. furiosus DSM 3638 has been
the source of several surprisingly flexible carbohydrate-active
enzymes that are stable at elevated temperatures.1b,1e,12 This
genome contains a gene (PF0589) annotated as a bifunctional
phosphomannose isomerase/GDP-mannose pyrophosphorylase
(manC) that catalyzes the first and third step of GDP-mannose
synthesis starting from fructose-6-phosphate (Scheme 1), but its
biochemical function has not yet been verified. Interestingly, the
recent biochemical characterization of the phosphomannomutase
from P. horikoshii OT3 uncovered its ability to act on not only
mannose-6-phosphate (2), but also glucose-6-phosphate.13 This
study further stimulated our curiosity as to the biochemical
function and potential utility of the manC gene. We report
herein the identification of a bifunctional PMI-GMP from the
hyperthermophile P. furiosus and its utility in the syntheses of a
number of activated mannoses and GDP-sugars.
Pseudomonas aeruginosa enzymes respectively (see ESI†). In our
previous studies we have reported that the hyperthermostable
archaeal enzymes possess broad substrate specificities with the
potential of expanding the synthesis of various natural and
non-natural sugar nucleotides for biotechnological applications.1e
However, those enzymes are not suitable for the synthesis of GDP-
activated sugars. To explore the value of the euryarchaea P. furiosus
genome as a reagent source for GDP-sugar syntheses, the 1286
base pair manC gene was cloned and expressed in Escherichia coli
R
BL21 Codon Plusꢀ (DE3)-RIPL strain. An approximately 53 kDa
protein, consistent with the molecular mass calculated from the
nucleotide sequence, was purified by simple Ni-affinity column and
was analyzed by SDS-PAGE (see ESI†). The resulting purified
protein did indeed have phosphomannose isomerase activity as
confirmed by a coupled assay that measures the formation of
NADPH at 340 nm.9b In addition to isomerase activity, the protein
could also produce GDP-mannose from mannose-1-phosphate
and GTP in the presence of inorganic pyrophosphatase (Scheme 1)
as monitored by an electrospray ionization mass spectrometry
(ESI-MS) assay,14 indicating that the manC is a bifunctional
enzyme. Although heat treatment should have denatured all native
proteins, in order to verify that the Ni-affinity purified protein
does not contain enzyme activity deriving from the E. coli itself,
an additional control experiment was run with extracts of E. coli
carrying a pET21a vector without an inserted gene. No additional
protein band corresponding to 53 kDa was detected by SDS-
PAGE analysis of the crude extract after passing through the Ni-
affinity column and no GDP-mannose pyrophosphorylase activity
was detected at 80 ◦C. No product formation was observed in the
absence of the enzyme, sugar phosphates, Mg2+ or NTP.
Since we hoped to find an effective, hyperthermostable version
of a GDP-sugar synthesizing biocatalyst, we focussed further
studies on the sugar nucleotidyltransferase activity of the enzyme.
Biochemical and kinetic properties of the enzyme were determined
by monitoring the formation of GDP-mannose from mannose-1-
phosphate and GTP. Because the recombinant protein was origi-
nally cloned from a hyperthermophilic archaeon, P. furiosus, which
grows optimally at 100 ◦C, the temperature dependence of the
enzymatic activity was analyzed between 0 and 100 ◦C. Maximum
activity of the purified manC was observed at 80 ◦C; however,
little loss of activity was detected at 99 ◦C even after 24 h of
incubation at this elevated temperature (Fig. 1A). The enzyme
remains stable for over 300 min without losing its activity when
stored at 80 ◦C. The recombinant protein exhibited relatively high
activity around pH 6.0 and 9.5, with a maximum in the range of
pH 7.0–8.0 in phosphate buffer (Fig. 1B).
Results and discussion
A
putative 463 amino acid residue of P. furiosus gene
product (PF0589) was annotated as mannose-6-phosphate
isomerase/mannose-1-phosphate guanyltransferase (manC) en-
zyme. BLAST search revealed that manC has a nucleotidyltrans-
ferase motif at the N-terminal site and a mannose-6-phosphate
isomerase (phosphomannose isomerase) motif at the C-terminal
site starting from residues 2 to 275 and 291 to 456, respectively.
The C-terminal domain has similarity with a type II phospho-
mannose isomerase which is generally a bifunctional enzyme. The
putative enzyme shares 44 and 47% identity with the E. coli and
It is well established that sugar nucleotidyl transferases require
divalent cations for their activity. To check the dependency of the
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the bifunctional manC is not specific to GTP; rather it accepts
all five major nucleoside triphosphates (Scheme 2). Next, the
tolerance of the enzyme to various sugar-1-phosphates with GTP
was tested. Relatively high activity was detected against glucose-,
galactose-, glucosamine-, N-acetylglucosamine- and fucose-1-
phosphate with the overall yield 30–85% (Scheme 2). In addition
to the GDP-sugars and NDP-mannoses, the enzyme is also
effective in the synthesis of NDP-glucoses and ADP-, CDP-,
UDP-GlcN (see ESI†). Overall, the enzyme can be used for
the synthesis of seventeen different nucleotide sugars from the
commercially available sugar 1-phosphates and NTPs. Based on
HPLC calculations, 85, 80, 70, and 45% yields were obtained
when the reactions were carried out on a 50 mg scale with
GTP and Man1P, Glc1P, GlcN1P and GlcNAc1P, respectively.
Interestingly, the enzyme did not have to be affinity purified
in order to convert the above mentioned sugar-1-phosphates to
their corresponding nucleotide sugars; the supernatant after heat
treatment of the recombinant E. coli extract worked as well as
the purified biocatalyst. This euryarchaeal manC clearly can turn
over a much greater range of substrates than the previously studied
crenarchaeal enzyme or enzymes (wild type or recombinant) from
mesophilic sources. For example, ATP, CTP and UTP were poor
substrates for the E. coli manC and only glucose-1-phosphate
and 2-deoxyglucose-1-phosphate could serve as alternate sugar-
1-phosphate substrates in low yields.9a Heat treatment of the
crude extract not only simplifies protein purification, but also
eliminates the possibility of contaminating E. coli proteins being
a source of alternate sugar nucleotidyltransferase activity. This
finding indicates that the archaeal sugar nucleotidyltransferase is
unusual in its ability to bind and turn over a range of nucleotide
triphosphate substrates.
Since the enzyme was shown to accept all 5 NTPs tested,
we were particularly interested in determining the effect of
substrate concentration on the enzymatic activity. The reaction
was performed with varying and fixed concentrations of Man1P
and NTP respectively as described above. Michaelis-Menten plots
of the velocity versus sugar-1-phosphate concentrations were
determined for the P. furiosus manC. Nonlinear regression analysis
of reactions run in triplicate using Man1P as the variable substrate
in the presence of fixed concentrations of ATP, CTP, GTP, dTTP
and UTP resulted in KM values of 120 8, 160 20, 72 7, 190
20, and 150 10 mM respectively (Table 1). As shown in Table 1,
the KM value obtained with Glc1P as the variable concentration in
the presence of fixed concentration of GTP was comparable to that
of Man1P. The results indicated that although the enzyme accepts
all NTPs, GTP and Man1P are the most preferred substrates of
the enzyme.
Fig. 1 Effect of temperature (A), pH (B) and Mg2+ ion concentration
(C) on the activity of the bifunctional manC. The enzyme (0.02 U) was
incubated with standard assay components at various temperatures for
10 min to determine the best temperature for activity. To check the pH
profile the enzyme (0.02 U) was incubated with standard assay components
at 80 ◦C for 10 min. The GDP-mannose formed was analyzed by an
ESI-MS based assay and expressed as the amount of GDP-mannose
formed after the reaction time. The optimum Mg2+ ion concentration was
found by monitoring the formation of GDP-mannose in the presence of
various concentrations of the divalent cation and the results expressed as
percentages of maximum activity.
enzyme on cations, various concentrations of divalent cations were
investigated in the forward reaction. Like other sugar nucleotidyl-
transferases the GDP-mannose pyrophosphorylase activity of the
bifunctional enzyme is absolutely dependent on divalent cations
with Mg2+ at 5 mM serving the best; however, the enzyme activity
remains high between 2–15 mM concentrations (Fig. 1C). The
order of effectiveness of the metal ions on the enzyme was shown
to be Mg2+ > Cu2+ > Zn2+ > Co2+ > Ca2+ and Mn2+.
Substrate promiscuity of a number of previously reported P.
furiosus enzymes from our group prompted us to verify the
specificity of manC with different substrates. Consequently the
substrate specificity of the enzyme was tested in the presence of
mannose-1-phosphate and various NTPs. The enzyme not only
accepts its natural substrate GTP, but also accepts ATP, CTP,
UTP and dTTP, which was confirmed by monitoring the formation
of ADP-man, CDP-man, UDP-man and dTDP-man using ESI-
MS. With the exception of dTDP-mannose, greater than 80% yield
was recorded in the syntheses of the four NDP-mannoses. These
results indicate that the sugar nucleotidyl transferase activity of
Because the enzyme has been shown to be highly effective
in the synthesis of a number of GDP-sugars and NDP-sugars
we decided to examine the role of the C-terminal domain in
the overall activity and substrate specificity of the N-terminal
nucleotidyltransferase domain. We created a truncated mutation
of the GMP by PCR amplification of the nucleotidyltransferase
domain gene and cloned and expressed it in E. coli. Substrate
specificity study of the truncated GMP showed that the mutant
accepts GTP and Man1P with about 100 fold lower GDP-man
pyrophosphorylase activity when the reaction was carried out
in the presence of Man1P and GTP (Table 1). However, unlike
the full length enzyme the truncated enzyme did not accept any
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Scheme 2 Synthesis of various sugar nucleotides. The conversion determined by ESI-MS was calculated as the difference between the amount of sugar
phosphate remaining in the reaction and the initial amount, then divided by the initial amount of substrate used in the reaction.1e
Table 1 Kinetic parameters of P. furiosus manC assayed with varying concentrations of sugar-1-phosphate and fixed concentrations of NTPs
Substrate
KM (mM)
Vmax (mM/min/mg)
k
cat/KM (mM-1 min-1)a
Man1P(3) (ATP)
3 (CTP)
120
160 20
8
11
11
50
1
1
2
11
8
3 (GTP)
72
7
90
62 9b
190 20
150 10
0.63 0.03b
0.81b
3 (TTP)
3 (UTP)
Glc1P (GTP)
6
14
14
1
1
1
4
11
18
94
7
a kcat was calculated based on the concentration of the purified enzyme. b indicates the kinetic parameters of the truncated enzyme.
other sugar-1P or NTPs. The temperature stability of the enzyme
remains the same as that of the full length enzyme. Likewise, the
mutant enzyme activity is absolutely dependent on divalent cations
with Mg2+ at 5 mM being the best. The pH profile of the truncated
enzyme remained the same as that of the wild type version.
These results indicate that the C-terminal domain is crucial for
the substrate promiscuity of the enzyme. The deletion of the
C-terminal domain may have changed the tertiary structure of the
enzyme; also, some key residues required for substrate promiscuity
might be missing. A related study found a similar phenomenon; a
thermostable truncated sugar-1-phosphate nucleotidyltransferase
(RmlA) from S. tokadaii strain 7 was shown to have 23 times
lower activity than that of the full length enzyme.11a This truncated
mutant also was shown to be inactivated at temperatures higher
than 60 ◦C.11a
prokaryotic and eukaryotic enzymes, the manC from P. furiosus
has an unusual tolerance of high temperatures thereby facilitat-
ing purification and storage of the recombinant protein. More
surprisingly, unlike previously reported enzymes that synthesize
GDP-mannose, this euryarchaeal sugar nucleotidyltransferase
can accept an unusually broad range of sugar phosphates and
sugar nucleotide triphosphates with reasonably efficient turnover
rates. This recombinant enzyme can provide various GDP-sugars
and NDP-mannoses without any costly and laborious protein
engineering approaches. The discovery of this stable, promiscuous
enzyme’s ability to provide analogs of GDP-mannose finally
enables detailed structure/function studies and possibly inhibition
of the pathways that build many of the oligosaccharide structures
necessary for pathogen virulence.
Acknowledgements
Conclusions
This material is based in part upon work supported by the National
Science Foundation under CAREER Grant No. 0349139. We
also acknowledge a Shimadzu University Research Grant and
a grant from the Herman Frasch Foundation, administered by
the American Chemical Society. N.L.B.P. is a Cottrell Scholar of
Research Corporation and an Alfred P. Sloan Research Fellow.
In summary, we have identified the first hyperthermostable, bi-
functional PMI-GMP from archaea which could be utilized for the
one-pot syntheses of a range of GDP-activated sugars and NDP-
mannoses. Although the general biochemical properties of this
euryarchaeal enzyme are similar to those of previously identified
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