Biochemical characterization of GDP-l-fucose de novo synthesis pathway in fungus Mortierella alpina

Article abstract of DOI:10.1016/j.bbrc.2009.12.116

Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. l-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-l-fucose (the nucleotide-activated form of l-fucose) in M. alpina. Genes encoding GDP-d-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-l-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k_m values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 °C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-l-fucose biosynthetic pathway in fungi.

Full text of DOI:10.1016/j.bbrc.2009.12.116

Biochemical and Biophysical Research Communications 391 (2010) 1663–1669
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Biochemical characterization of GDP-L-fucose de novo synthesis pathway
in fungus Mortierella alpina
a
c
a
b
c
a
b,*
Yan Ren , Andrei V. Perepelov , Haiyan Wang , Hao Zhang , Yuriy A. Knirel , Lei Wang , Wei Chen
a
TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457, PR China
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow, Russian Federation
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 December 2009
Available online 24 December 2009
Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount
of polyunsaturated fatty acids. -Fucose is an important sugar found in a diverse range of organisms, play-
ing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-
-fucose (the nucleotide-activated form of -fucose) in M. alpina. Genes encoding GDP- -mannose 4,6-
dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed
heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion pro-
teins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy
L
L
L
D
Keywords:
Mortierella alpina
GDP-
Fucosylation
GDP- -mannose 4,6-dehydratase
GDP-keto-6-deoxymannose 3,5-epimerase/
-reductase
L-fucose
mannose to GDP-
tion-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k
GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and
.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum
L
-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ioniza-
D
m
values of GMD for
4
1
temperature and pH were determined to be 37 °C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations
are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT.
To our knowledge this is the first report on the characterization of GDP-
fungi.
L-fucose biosynthetic pathway in
Ó 2009 Elsevier Inc. All rights reserved.
Introduction
-Fucose (6-deoxy-
found in both prokaryotes and eukaryotes. It is a sugar component
of bacterial lipopolysaccharides [1], and cell wall polysaccharides
the substrate. GDP-
nose via a de novo pathway or from
The salvage pathway, operated by fucose kinase (FUK) and GDP fu-
cose pyrophosphorylase (GFPP), is mostly found in animals and
L
-fucose may be synthesized from GDP-
D-man-
L-fucose in a salvage pathway.
L
L
-galactose) is an important monosaccharide
plants, and is mainly used for the recycling of
cellular fucose-containing macromolecules [6]. In the de novo path-
way (Fig. 1), GDP- -mannose is converted to GDP-4-keto-6-deoxy-
-mannose by GDP- -mannose 4,6-dehydratase (GMD) and then to
-fucose by GDP-keto-6-deoxymannose 3,5-epimerase/4-
L-fucose derived from
in plants [2] and some fungi of specific taxa [3–5]. L-Fucose is also
a common component of sugar moiety in glycoconjugates and fre-
quently exists as a terminal glycosylation residue [6]. Glycoconju-
gates including glycoproteins and glycolipids are known to play
many important biological roles such as recognition and signaling,
protein folding and conformation, and membrane stabilization [7–
D
D
D
GDP-
L
reductase (GMER), a bifunctional enzyme with epimerase and
reductase activities [6]. The function of GMD and GMER from Homo
sapiens [9–10], Helicobacter pylori [11], Escherichia coli [12–13],
Caenorhabditis elegans and Drosophila melanogaster [14], Parame-
cium bursaria Chlorella Virus [15] and Arabidopsis thaliana [16] have
been characterized in vitro.
8
].
Fucosylation is carried out by fucosyltransferases, which re-
quire the activated nucleotide form of -fucose (GDP- -fucose) as
L
L
Mortierella alpina is a well-known polyunsaturated fatty acids
PUFA)-producing oleaginous fungus commonly found in soil,
and is one of industrial species for PUFA production [17]. Recently,
we have sequenced the whole genome of M. alpina (ATCC 32222)
Abbreviations: GMD, GDP-D-mannose 4,6-dehydratase; GMER, GDP-keto-6-
(
deoxymannose 3,5-epimerase/4-reductase; COSY, correlation spectroscopy; HSQC,
heteronuclear single-quantum coherence; HMQC, heteronuclear multi-quantum
coherence; TOCSY, total correlation spectroscopy
Corresponding author. Address: State Key Laboratory of Food Science and
Technology, School of Food Science and Technology, Jiangnan University, Wuxi,
*
(
separate study). Sequence analysis reveals the presence of the de
novo pathway for the synthesis of GDP- -fucose in M. alpina. In this
study, we characterized the function of GMD and GMER from M.
L
1
800 Lihu Ave. Wuxi, Jiangsu 214122, PR China. Fax: +86 510 85912155.
E-mail address: weichen@jiangnan.edu.cn (W. Chen).
0
006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.12.116

1
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Y. Ren et al. / Biochemical and Biophysical Research Communications 391 (2010) 1663–1669
Fig. 1. De novo (A) and salvage (B) synthesis pathways of GDP-L-fucose.
alpina, in vitro. Kinetic parameters and some other properties of
GMD and GMER were examined. Comparative analyses of the M.
alpina GMD and GMER proteins with other homologous proteins
tography with a Chelating Sepharose Fast Flow column (GE Health-
care) according to the manufacturer’s instruction. Unbound
proteins were washed out with 100 ml of wash buffer (50 mM
Tris–HCl, pH 8.0, 300 mM NaCl, and 25 mM imidazole). Fusion pro-
teins were eluted with 3 ml of elution buffer (50 mM Tris–HCl, pH
were carried out, and physiological role of
are discussed.
L-fucose in M. alpina
8
.0, 300 mM NaCl, and 250 mM imidazole), and dialyzed overnight
against 50 mM Tris–HCl buffer containing 20% glycerol (pH 7.4) at
°C. Protein concentration was determined by the Bradford meth-
Materials and methods
4
od. Purified proteins were stored at ꢁ80 °C.
Strains and growth conditions. Mortierella alpina (American Type
Culture Collection Catalog No. 32222) was cultured on potato dex-
trose agar at 25 °C for 5–7 days. When the growth was visible, the
culture was inoculated into 200 ml of the medium containing
Enzyme activity assays. The reaction mixture for GMD contained
5
1
0 mM Tris–HCl (pH 8.0), 1 mM GDP-
D
-mannose (Sigma–Aldrich),
.5 mM NAD or NADP (Sigma–Aldrich), 1 M of purified GMD, in
l. The reaction mixture for GMER contained
l of the GMD reaction mixture to supply the substrate for
GMER, 50 mM Tris–HCl (pH 8.0), 2.5 mM NADH or NADPH (Sig-
ma–Aldrich), 0.15 M of purified GMER, in a total volume of
l. Reactions were carried out at 37 °C for 30 min unless other-
+
+
l
a total volume of 10
10 l
l
1
3 2 4
00 g/L glucose, 5 g/L yeast extract, 3 g/L NaNO , 1 g/L KH PO ,
and 0.5 g/L MgSO O, and incubated at 25 °C for 4 days with
4
ꢀ7H
2
shaking. The mycelia were collected by filtration through 10 layers
of sterile cheesecloth, and immediately frozen into liquid nitrogen.
Cloning and plasmid construction. Total RNA was isolated using
Trizol reagent (Invitrogen). The genes encoding GMD and GMER
were cloned by RT-PCR. Reverse-transcription of mRNA into cDNA
was carried out with oligo(dT) primer using Superscript II reverse
transcriptase (Invitrogen). PCR was performed with the primer pair
l
20 l
wise specified before terminated by adding equal volume of chlo-
roform. GMD and GMER products were collected by centrifugation
for 5 min at 12000 rpm on an Eppendorf Centrifuge 5810R, and
subjected to CE, ESI-MS and NMR analysis. Enzyme activity was
indicated by the conversion of the substrate to the product.
CE analysis. Capillary electrophoresis (CE) was performed using
a Beckman Coulter P/ACE MDQ Capillary Electrophoresis System
equipped with a PDA detector (Beckman Coulter, CA). The capillary
0
0
GCGAATTCATGTCTTCTCCTATTGAG-3 /5 -CTGAAGCTTTTAGTTGAA-
0
0
0
0
GACATCG-3 (GMD) or 5 -CAGAATTCATGTCTCCCTCAAAGTC-3 /5 -
0
CATAAGCTTTTACTTGCGGATGGT-3 (GMER) (restriction sites
underlined), and the PCR condition used was: denaturation at
was bare silica of 75
capillary was conditioned before each run by washing with 0.1 M
l
m ꢂ 57 cm, with the detector at 50 cm. The
9
5 °C for 30 s, annealing at 55 °C for 45 s and extension at 72 °C
for 2 min, 25 cycles, in a final volume of 50 l. The amplified prod-
l
+
NaOH first, and then 25 mM borate-sodium hydroxide (pH 10.0)
ucts were cloned into pET28a to construct pLW1534 (containing
GMD) or pLW1535 (containing GMER). Presence of the inserts in
the plasmids was confirmed by sequencing using an ABI 3730
Sequencer.
(
used as the mobile phase) for 5 min each. Samples were loaded
by pressure injection at 0.5 p.s.i. for 10 s, and electrophoresis
was carried out at 20 kV. Peak integration and trace alignment
were performed with the Beckman P/ACE station software (32 Kar-
at versions 5.0). Conversion ratio was calculated from peak areas of
substrate and product.
Reverse-phase HPLC and ESI-MS analysis. The GMD and GMER
reaction product were purified by reverse-phase HPLC using a Bio-
CAD 700E Perfusion Chromatography Workstation (Applied Bio-
Protein expression and purification. Escherichia coli BL21 (DE3)
carrying pLW1534 or pLW1535 was grown in LB medium contain-
ing 50 lg/ml kanamycin overnight at 37 °C with shaking. The over-
night culture was inoculated into 500 ml of fresh medium and
grown to OD600 0.6. Expression of GMD was induced by 1 mM IPTG
at 25 °C for 4 h, and expression of GMER was induced by 0.05 mM
IPTG at 16 °C for 5 h. After the IPTG induction, cells were harvested
by centrifugation, washed with binding buffer (50 mM Tris–HCl,
pH 8.0, 300 mM NaCl, and 10 mM imidazole), resuspended in the
same buffer containing 1 mM phenylmethanesulfonyl fluoride
and 1 mg/ml lysozyme, and sonicated (Ultraschallprozessor
UP200S). The cell debris was removed by centrifugation, and the
systems, CA) with a Venusil MP-C18 column (5 lm particle,
4
.6 mm ꢂ 250 mm) (Agela Technologies). The mobile phase used
was composed of 4% of acetonitrile and 96% of 50 mM triethyl-
amineacetatic acid (pH 6.8). The flow rate was 0.6 ml/min. Frac-
tions containing expected products were collected, lyophilized
and re-dissolved in methanol before injecting into a Finnigan
LCQ Advantage MAX ion trap mass spectrometer (Thermo Electron,
CA) at negative mode (4.5 kV, 250 °C) for ESI-MS analysis. For MS2
supernatant containing soluble proteins was collected. The His
6
-
tagged fusion proteins were purified by nickel ion affinity chroma-

Y. Ren et al. / Biochemical and Biophysical Research Communications 391 (2010) 1663–1669
1665
and MS3 analyses, nitrogen was used as collision gas and helium as
auxiliary gas and collision energies used were typically 20–30 eV.
NMR spectroscopy and chemical analysis. A sample of purified
5 min in a final volume of 20
l
l. Conversion of GDP-mannose to
GDP-4-keto-6-deoxy mannose, and GDP-4-keto-6-deoxy mannose
to GDP- -fucose was monitored by CE analysis. k and Vmax values
L
m
GDP-b-
from 99.9% H
L
-Fucp (0.2 mg) was deuterium-exchanged by freeze-drying
were calculated based on the Michaelis–Menten equation.
2
2
2
O, dissolved in 99.96% H
2
O (150
lL) and examined
Determination of temperature and pH optima, divalent cations
requirements and protein reducing reagent effect. To determine the
temperature optima for GMD and GMER, reactions were carried
out at different temperatures (15, 25, 37, 50, and 55 °C), pH 8.0,
for 15 min. To determine the pH optima, reactions were carried
out at different pH values (pH 5–12), 37 °C, for 15 min. To test
the effects of divalent cations and protein reducing reagent on en-
zyme activity, reactions were carried out in the presence of 5 mM
using a Shigemi (Japan) microtube. NMR spectra were recorded on
a Avance 600 NMR spectrometer (Bruker, Germany) at 30 °C using
internal sodium trimethylsilyl-[2,2,3,3–2H4] propanoate (dH 0.00),
3 4
acetone (dC 31.45) and external aqueous 85% H PO (dP 0) as refer-
ences. Two-dimensional NMR spectra were obtained using stan-
dard pulse sequences from the manufacturer, and TopSpin
program (Bruker) was used to acquire and process the NMR data.
GDP-b-
100 °C, 2 h) and
S)-2-octyl glycosides [18] using a Hewlett–Packard 5880 instru-
L
-Fucp was hydrolyzed with 2 M trifluoroacetic acid
of MgCl
for 10 min.
Nucleotide sequence accession numbers. The complete gene se-
quences of GDP- -mannose 4,6-dehydratase (GMD) and GDP-
keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) have
been deposited at GenBank under the accession numbers
GU299800 and GU299801.
2 2 2 2 2
, MnCl , FeCl2, CuCl , CaCl , CoCl , or DTT at 37 °C, pH 8.0,
(
(
L
-fucose was identified by GLC of the acetylated
ment equipped with an Ultra-2 column (0.20 mm ꢂ 25 m) and a
D
temperature program of 160 (3 min) to 290 °C at 3 °C/min.
Measurement of kinetic parameters. To measure the k
m
and Vmax
value of GMD for GDP-mannose, reactions were carried out with
various concentrations of GDP-mannose (0.4–1.5 mM), and fixed
+
concentration of NAD (2 mM) and GMD (1.13
l
M). To measure
Results
m
the k and Vmax value of GMER for GDP-4-keto-6-deoxy mannose,
reactions were carried out with various concentrations of GDP-4-
keto-6-deoxy mannose (0.5–1.5 mM), and fixed concentration of
Expression and purification of GMD and GMER
NADPH (2.5 mM) and GMER (0.10
m
lM). To measure the k and
GMD and GMER from Mortierella alpina were expressed as His-
tagged fusion proteins in E. coli BL21 by IPTG induction, and puri-
fied to near homogeneity by nickel ion affinity chromatography
Vmax values of GMER for NADH and NADPH, reactions were carried
out with various concentrations of NADH or NADPH (0.3–2 mM)
and fixed concentration of GDP-4-keto-6-deoxy mannose (1 mM)
and GMER (0.17 lM). All reactions were carried out at 37 °C for
(Sup. Fig. 1A). The molecular masses estimated by SDS–PAGE anal-
yses were 45 kDa for GMD and 40 kDa for GMER, corresponding
Fig. 2. CE chromatographs of GMD and GMER reaction products. Shown are GDP-
D
-mannose standard (A); NAD+ standard (B); GMD reaction without GMD (C); GMD reaction
with GMD (D); NADH standard (E); GMER reaction without GMER (F); and GMER reaction with GMER (G).

1
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Y. Ren et al. / Biochemical and Biophysical Research Communications 391 (2010) 1663–1669
well to the calculated masses (41.2 kDa and 36.4 kDa plus His tag).
The molecular masses estimated by native-PAGE were 140 Kb for
GMD and 232 Kb for GMER, indicative of a trimeric and a hexa-
meric protein, respectively (Sup. Fig. 1B).
Analysis of GMD and GMER products by electro-spray ionization–mass
spectrometry
The GMD (the 14.1 min peak) and GMER (the 13.2 min peak)
products were purified by reverse-phase HPLC (data not shown),
and analyzed by ESI-MS. Ion peaks at m/z 586.10 from the GMD
product and m/z 588.19 from the GMER product were obtained,
corresponding well to the masses for GDP-4-keto-6-deoxyman-
nose (m/z 586) and GDP-fucose (m/z 588), respectively [16]. MS/
MS (MS2) analysis of the two ion peaks (586.10 and 588.19) and
the follow-up MS/MS/MS (MS3) analysis of selected MS2 ion peak
(442.0 and 442.06) resulted in the detection of ion peaks matching
the fragments derived from GDP-4-keto-6-deoxy mannose and
GDP-fucose, respectively (Sup. Fig. 2A). Fragments corresponding
to each peak are depicted in Sup. Table 1.
CE analysis of GMD and GMER products
As shown in CE chromatographs (Fig. 2), GDP-mannose identi-
fied as the peak eluted at 12.8 min was converted to a peak eluted
at 14.1 min in the reaction catalyzed by GMD in the presence of
+
NAD as the electrons and hydrogen ions carrier (Fig. 2). The same
+
+
result was obtained when NADP was used instead of NAD (data
not shown). In the reaction catalyzed by GMER, the 14.1 min peak
(
GMD product) was converted to a new peak eluted at 13.2 min,
+
accompanied by the conversion of NADH to NAD as judged by in-
creased NAD /NADH ratio (Fig. 2), and the same was observed
+
when NADPH was used instead of NADH (data not shown). There-
fore, GMER possesses NAD(P)H-dependent reductase activity. In
either reaction catalyzed by GMD or GMER, 100% of the conversa-
tion rate could be achieved.
Analysis of GMER product by NMR
The identity of the GMER product as GDP-
ally confirmed by NMR analysis. Fucose was identified and its
configuration was confirmed by GLC analysis of the acetylated
S)-2-octyl glycosides, which were derived after full acid hydrolysis
of GDP-b- -Fucp.
A sample of GDP-b-
L-fucose was structur-
L-
(
Table 1
L
1
H and 13C NMR chemical shifts of GDP-b-L-Fucp (d, ppm). The H8/C8 correlation of
L-Fuc was studied by two-dimensional shift-
guanine is at d 8.1/138.5.
correlated 1H, 1H, 1H, 13C and 1H, 31P NMR spectroscopy. The 1H
NMR chemical shifts were assigned using 1H, 1H COSY, and TOCSY
experiments (Table 1). In the TOCSY spectrum there were correla-
tions of H1 with H2–H4 and H5 with H6 of fucopyranose; H1’ with
H2’–H4’ and H3’ with H4’ and H5’ of ribofuranose. Coupling con-
stant values determined from the 1H NMR spectrum, including
J1,2 7.8 and J4,5 < 1 Hz, confirmed the b-galacto configuration of
Sugar residue
Nucleus
Atom
1
2
3
4
5
6
1
1
1
1
b-Fucp
b-Ribf
H
C
4.92
98.8
5.93
88.1
3.56
72.4
4.81
74.7
3.65
73.9
4.54
71.9
3.70
72.5
4.35
84.2
3.76
72.8
4.21 (2H)
66.7
1.23
16.8
3
H
3
C
Fig. 3. (A) Parts of a 1H,13C HSQC spectrum, (B) 1H,31P HMQC spectrum, and (C) structure of GDP-b-L-Fucp. Arabic numerals refer to atoms in Fuc and GDP designated as F
and G, respectively. Signals of contaminating substances from HPLC buffer are marked with asterisk.

Y. Ren et al. / Biochemical and Biophysical Research Communications 391 (2010) 1663–1669
1667
the pyranose sugar, which is thus b-Fucp. A 1H,13C HSQC experi-
ment (Fig. 3A) revealed proton-to-carbon correlations for Fuc, Rib
and guanine, and the assigned 13C NMR chemical shifts (Table 1)
were in good agreement with published data for GDP [19] and b-
Fucp [20]. The 31P NMR spectrum contained signals for a diphos-
phate group at -10.9 and -12.7, which showed correlations with
H5’ of GDP at -10.9/5.93 and H1 of b-Fucp at -12.7/4.92 in the
culation of kinetic parameters. The kinetics of the reactions
catalyzed by both GMD and GMER fit well to the Michaelis–Men-
ten model, and the kinetic parameters are listed in Table 2. GMER
showed similar affinities to NADH and NADPH, but the Vmax value
was higher for NADPH (0.024 versus 0.012 mM/min). Therefore,
NADPH is a better coenzyme for GMER.
1
H,31P HMQC spectrum (Fig. 3B). These data together confirmed
Effects of temperature, pH, divalent cations and protein reducing
reagent on the activity of GMD and GMER
finally the structure of GDP-b- -Fucp as depicted in (Fig. 3C).
L
Kinetic parameters of GMD and GMER
GMD and GMER activities were detected at 15 to 55 °C and pH
5
.0 to 11.0 (Fig. 4A & B). The optimum temperature was 37 °C for
Kinetic parameters of GMD (for GDP-mannose) and GMER (for
GDP-4-keto-6-deoxy mannose, NADH and NADPH) were mea-
sured. The initial velocities were determined and used for the cal-
both enzymes, and the optimum pH was pH 9.0 for GMD or pH
2+
7.0 for GMER. GMD activity was significantly enhanced by Mg
,
2
+
2+
2+
2+
strongly inhibited by Fe , Cu and Co , but not affected by Ca
Table 2
Kinetics parameters. The data shown are the average from three independent experiments.
Enzyme
Substrate
GDP- -mannose
GDP-4-keto-6-deoxy-
NADPH
K
m
(mM)
V
max (mM/min)
K
cat/min
GMD
D
0. 77 ± 0.007
1.047 ± 0.015
1.025 ± 0.054
1.157 ± 0.089
0.041 ± 0.002
0.033 ± 0.002
0.024 ± 0.002
0.012 ± 0.002
357.71 ± 18.73
GMER
GMER
GMER
D-mannose
1664.72 ± 106.08
698.89 ± 58.24
334.89 ± 61.77
NADH
Fig. 4. Effects of temperature (A), pH (B), divalent cations and DTT (C) on the activities of GMD and GMER. 100% GMD activity represents 0.59 (A), or 0.78 (B), or 0.58 enzyme
unit (C). 100% GMER activity represents 0.98 (A), 1.15 (B), or 0.81 enzyme unit (C). One enzyme unit is defined as the amount of purified enzyme (mg) required for the
conversion of 100
lmol GDP-mannose to GDP-4-keto-6-deoxymannose (GMD), or for the conversion of 200 lmol GDP-4-keto-6-deoxymannose to GDP-fucose (GMER) per
hour. The data shown are the average from three independent experiments.

1
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Y. Ren et al. / Biochemical and Biophysical Research Communications 391 (2010) 1663–1669
and Mn2+. GMER activity was slightly enhanced by Ca and Mg2+
,
2+
duction of glycolipids such as cerebrosides [29]. It is speculated
that fucosylation of proteins and/or lipids may also occur in M. alp-
ina, and play important cellular roles, especially in lipid
metabolism.
2+
completely inhibited by Cu , and inhibited to different degrees by
2
+
2+
2+
Co , Fe , and Mn (Fig. 4C). DTT enhanced GMD activity by 2.3
folds and GMER activity by 2.1 folds (Fig. 4C). Enhancement of
GMD and GMER activity by DTT was also reported for other char-
acterized homologous proteins such as Helicobacter pylori enzymes
To the best of our knowledge, this is the first report on the char-
acterization of the GDP-L-fucose biosynthesis pathway in fungi.
[
11].
The role of
investigated.
L-fucose in M. alpina is worthy to be further
Discussion
GMD shares 54 to 72% identities to other characterized GMD
Acknowledgements
We thank Professor A.S. Shashkov for help with NMR spectros-
copy. This work was supported by the NSFC General Program
Grants 30870070, the National 863 program of China grants
2006AA02Z106, the Research Program of State Key Laboratory of
Food Science and Technology (SKLF-MB-200802), the National Sci-
ence Foundation of China (NSFC) Key Program Grants 20836003,
and the Russian Foundation for Basic Research (grant 08-04-
01205 to A.V.P.).
proteins, and is most similar to Homo sapiens GMD. Multiple se-
quence alignment of M. alpina GMD and other characterized
GMD proteins reveals the presence of all conserved motifs and res-
idues described for GMD in M. alpina GMD (Sup. Fig. 3A), including
a Gly-X-X-Gly-X-X-Gly motif at the N terminus involved in co-fac-
tor binding [21], 7 conserved residues at C terminus (Asn201,
Val212, Lys215, Arg240, Leu233, Val274, and Tyr315) involved in
sugar-nucleotide binding [21], Thr148-Tyr172-Lys176 catalytic
triad, and Glu150 at active-site [21]. GMER shares 27 to 61% iden-
tity to other characterized GMER proteins, and is most similar to D.
melanogaster GMER. Multiple sequence alignment of the GMER
proteins reveals the presence of all conserved motifs and residues
described for GMER in M. alpina GMER (Sup. Fig. 3B), including Gly-
X-X-Gly-X-X-Gly motif at the N terminus for co-factor binding
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2009.12.116.
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