Báo cáo khoa học: Functional expression of Pseudomonas aeruginosa GDP-4-keto6-deoxy-D-mannose reductase which synthesizes GDP-rhamnose docx

Functional expression of
Pseudomonas aeruginosa
GDP-4-keto-
6-deoxy-
D
-mannose reductase which synthesizes GDP-rhamnose
Minna MaÈki
1
, Nina JaÈ rvinen
1
, Jarkko RaÈ binaÈ
1
, Christophe Roos
2
, Hannu Maaheimo
3
,
Pirkko Mattila
2
and Risto Renkonen
1
1
Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, University of Helsinki, Finland;
2
MediCel, Helsinki, Finland;
3
VTT Biotechnology, Espoo, Finland;
4
HUCH Laboratory Diagnostics,
Helsinki University Central Hospital, Helsinki, Finland
Pseudomonas a eruginosa is an opportunis tic Gram-negative

bacterium that c auses severe infections in a number of hosts
from plants to mammals . A-band lipopolysaccharide of
P. aeruginosa contains
D
-rhamnosylated O-antigen. The
synthesis of GDP-
D
-rhamnose, the
D
-rhamnose donor in
D
-rhamnosylation, starts from GDP-
D
-mannose. It is ®rst
converted by the GDP-mannose-4,6-dehydratase (GMD)
into GDP-4-keto-6-deoxy-
D
-mannose, and t hen redu ced to
GDP-
D
-rhamnose by GDP-4-keto-6-deoxy-
D
-mannose
reductase (RMD). Here, we describe the enzymatic c har-
acterization of P. aeruginosa RMD expressed in Sacchar-
omyces cerevisiae. Previous success in functional expression
of bacterial gm d genes i n S. cerevisiae allowedustoconvert
GDP-
D
-mannose into GDP-4-keto-6-deoxy-

D
-mannose.
Thus, coexpression of the Helicobacter pylori gmd and
P. aeruginosa rmd genes resulted in conversion of the
4-keto-6-deoxy intermediate into GDP-deoxyhexose. This
synthesized GDP-deoxyhexose was con®rmed t o be GDP-
rhamnose by HPLC, matrix-assisted laser desorption/ion-
ization time-of-¯ight MS, and ®nally NMR spectroscopy.
The functional expression of P. aeruginosa RMD in
S. cerevisiae will provide a tool for generating GDP-rham-
nose f or in vitro rhamnosylation of g lycoprotein a nd glyco-
peptides.
Keywords: A-band O-antigen; GDP-4-keto-6-deoxy-
D
-mannose r eductase(RMD);GDP-rhamnose; Pseudomonas
aeruginosa.
Pseudomonas a eruginosa is an opportunis tic Gram-negative
bacterium t hat c an cause infections in immunocompro-
mised patients including th ose with severe burn wounds,
cystic ®brosis and c ancer. The lipopolysaccharides that are
cell surface m olecules and virulence fac tors of P. aeruginosa
are both endotoxic and protective against serum-mediated
lysis. The latter phenomenon is mainly due to the highly
heterogeneous O-antigen (O-polysaccharide). P. aeruginosa
synthesizes concomitantly two chemically distinct variants
of lipopolysaccharide, designated A and B bands [1]. The
A-band O-antigen is a homopolymer consisting of
D
-rhamnose s ugar residues arranged as repeating trisaccha-
ride units ()3

D
-Rhaa1±2
D
-Rhaa1±3
D
-Rhaa1-)
n
[2]. In con-
trast, the B-band O-antigen is a heteropolymer composed of
repeating disaccharide to pentasaccharide units of many
different monosaccharides [3].
Rhamnose is a deoxyhexose sugar found widely in
bacteria and plants, but not in mammals. Of the two
isomers,
L
and
D
, the former is much more common. The
L
-isomer is found from the core oligosaccharide, B-band
O-antigen and rhamnolipids of P. aeruginosa, while the
D
-isomer is found from the A-band O-antigen [1,4]. The
route of synthesis of GDP-
D
-rhamnose, the precursor of
D
-rhamnosylated glycans, was proposed in the 1960s [5]. It
starts from GDP-
D

-mannose, which is ®rst converted into
GDP-4-keto-6-deoxy-
D
-mannose by GDP-mannose-4,6-
dehydratase (GMD) (Fig . 1). This 4-keto-6-deoxy interme-
diate can be reduced to the GDP-monodeoxyhexose,
GDP-
L
-fucose, GDP-
D
-rhamnose, GDP-deoxy-
D
-talose
or GDP-deoxy-
D
-altrose, by separate enzymes (in the case
of GDP-
L
-fucose th e 3,5-epimerization occurs before
reduction). It is also an intermediate in the conversion of
GDP-
D
-mannose into the GDP-dideoxyhexose, GDP-col-
itose [6], and the GDP-dideoxy amino sugar, GDP-
D
-per-
osamine [ 7]. In the pathway o f GDP-
D
-rhamnose synthesis,
the GDP-4-keto-6-deoxy-

D
-mannose reductase (RMD) is
responsible for the targeted reduction of the 4-keto group,
with NADH and NADPH as hydride donors [8].
The genetics of O-antigen biosynthesis in P. aeruginosa
has been extensively studied by Rocchetta et al.[1].On
the basis of mutagenesis analyses, it was proposed that the
function of the rmd gene could be the conversion of the
4-keto-6-deoxy intermediate into GDP-
D
-rhamnose [9].
However, the rmd gene has not been expressed and therefore
its enzymatic properties have not been characterized.
The aims of this study were to provide proof of the
function of the P. aeruginosa rmd geneandtosynthesize
GDP-rhamnose for further glycobiological use. We have
previously shown that Saccharomyces cerevisiae is an ideal
host for expressing enzymes needed for the synthesis of
Correspondence to R. Renkonen, Department of Bacteriology and
Immunology, Haartman Institute and Biomedicum, PO Box63,
FIN-00014 University of Helsinki, Helsinki, Finland.
Fax: + 359 9 19125110, Tel.: + 359 9 19125111,
E-mail:
Abbreviations: GMD, GDP-mannose-4,6-dehydratase; RMD, GDP-
4-keto-6-deoxy-
D
-mannose reductase; MALDI-TOF, matrix-assisted
laser desorption/ionization time-of-¯ight.
(Received 4 October 2001, accepted 19 November 2001)
Eur. J. Biochem. 269, 593±601 (2002) Ó FEBS 2002

deoxyhexose sugar nucleotides [10] because the glycosyla-
tion is largely restricted to mann osylation and yeast is not
known to have deoxyhexose metabolism of its own [11,12].
Therefore, the cytoplasm of yeast cells is a relative ly rich
sourc e of GD P-
D
-mannose, the starting material for the
rhamnose p athway, without any competing e ndogenous
enzymatic activity. As detection and identi®cation of
various deoxyhexoses is not straightforward, the lysates of
yeast transformants were analyzed by HPLC, matrix-
assisted laser desorption/ionization time-of-¯ight ( MAL-
DI-TOF) MS and
1
H NMR to verify the structure of the
synthesized GDP-rhamnose.
EXPERIMENTAL PROCEDURES
Strains and culture conditions
The bacterial and yeast strains and plasmids used in this
study are listed in Table 1. P. aeruginosa and Escherichia
coli were grown at 37 °C, and t he media used for bacterial
culture and maintenance were King's broth [13] and Luria
broth [14], respectively. S. cerevisiae strains were grown at
30 °C. For the S. cerevisiae host strains, the medium for
culture and maintenance was YPAD medium [14], and for
strains harboring pESC-LEU plasmid or its derivatives, the
medium was synthetic dextrose minimal medium (SD
dropout medium) lacking leucine [14]. Synthetic galactose
minimal medium (SG dropout medium) lacking leucine [14]
was used for the fusion protein inductions. When appro-

priate, antibiotic concentrations for plasmid propagation
were 50 lgámL
)1
kanamycin and 100 lgámL
)1
ampicillin.
Recombinant DNA techniques
Chromosomal DNA was isolated from P. aeruginosa
ATCC 27853 using a QIAamp Tissue kit (Qiagen, Hilden,
Germany). The rmd gene was a mpli®ed from chromosomal
DNA u sing the primer set RMDF 5¢-GAAGATCTTA
ACTCAGCGTCTGTTCGTC (creating a BglII site) and
RMDR 5¢-GGTTAATTAATCAGATAAAAGGCCCG
CTT (creating a PacI site). The PCR product was ®rst
cloned into the pCR-XL-TOPO vector using the TOPO XL
Cloning kit (Invitrogen, Carlsbad, CA, USA). The rmd gene
was digested out and subcloned into the BglII/PacIsites
of the pESC-LEU (Stratagene, La Jolla, CA, USA) and
the pHP1 (N. Ja
È
rvinen, M. Ma
È
ki, J. Ra
È
bina
È
, C. Roos,
P. Mattila & R. Renkonen, unpublished) vectors in-frame
with an N-terminal FLAG epitope, yielding the corre-
sponding plasmids pRHA1 and pRHA2. pHP1 is a

derivative of the pESC-LEU vector containing the H. pylori
gmd gene cloned in-frame with an N-terminal c-Myc epitope
Table 1. Bacterial and yeast strains and plasmids.
Strain/plasmid Description Reference
Strain
P. aeruginosa
ATCC 27853 ATCC
E. coli
TOP10 F
±
mcrA D(mrr-hsdRMS-mcrBC) /80lacZDM15
DlacX74 deoR recA1 araD139 D(ara-leu)7697
galU galK rpsL(Str
R
) endA1 nupG
Invitrogen
S. cerevisiae
YPH501 ura3±52 lys2±801
amber
ade2±101
ochre
trp1-D63
his3-D200 leu2-D1, mating type a/a
Stratagene
Plasmid
pCR-XL-TOPO E. coli cloning vector Invitrogen
pESC-LEU S. cerevisiae expression vector Stratagene
pHP1 pESC-LEU derivate containing the H. pylori
gmd gene as a 1143-bp fragment under GAL10 promoter
N. Ja

È
rvinen
et al. (unpublished)
pRHA1 pESC-LEU derivate containing the P. aeruginosa
rmd gene as a 915-bp fragment under GAL1 promoter
This study
pRHA2 pESC-LEU derivate containing the H. pylori gmd gene
under GAL10 promoter and the P. aeruginosa rmd gene
under GAL1 promoter
This study
Fig. 1. Biosynthetic pathways of the deoxyhexoses from the common
4-keto-6-deoxy intermediate. GDP-
D
-mannose is ®rst converted into
the 4-keto-6-deoxy intermediate, which is then reduced to dierent
deoxyhexoses. The enzymes involved in the GDP-
D
-fucose and GDP-
D
-rhamnose pathways have been characterized, whereas the reaction
steps and enzymes involved in the GDP-deoxy-
D
-talose and GDP-
deoxy-
D
-altrose pathways have not been identi®ed.
594 M. Ma
È
ki et al.(Eur. J. Biochem. 269) Ó FEBS 2002
(N. Ja

È
rvinen, M. Ma
È
ki, J. Ra
È
bina
È
,C.Roos,P.Mattila&
R. Renkonen, unpublished). The recombinant plasmids
were sequenced on an automated A BI 3100 sequencer (PE
Biosystems). pESC-LEU, pHP1, pRHA1 and pRHA2
vectors were transformed into S. cerevisiae host strains by
the lithium acetate method following the instructions of the
manufacturer (Stratagene). The transformants were selected
on the SD dropout plates lacking leucine.
Protein expression and analysis
The GAL1 and GAL10 promoters of the pESC-LEU
expression vector are repressed by dextro se and induced by
galactose. In the expression experiments, the yeast strains
were ®rst grown in the SD dropout medium overnight at
30 °C. After centrifugation, overnight cultures were inocu-
lated into the same volume of the SG dropout medium and
then incubated for 24 h at 3 0 °C. The yeast cells w ere
harvested and ly sed with Y -PER Yeast Protein Extract ion
reagent (2.5 mL for 1 g cell paste; Pierce, Rockford, IL,
USA) supplemented with 5 m
M
MgCl
2
,200l

M
GDP-
D
-mannose (Sigma, St Louis, MO, USA), 200 l
M
NADP
+
(Calbiochem, San Diego, CA, USA) and 200 l
M
NADPH
(Calbiochem). The suspensions were agitated gently for
20 min at room temperature and the cell debris removed by
centrifugation at 13 000 g for 10 min. The cell lysates were
assayed for protein expression as well as enzyme activity.
Expression of the fusion proteins was analyzed by Western
blot u sing antibodies (Invitrogen) against the c-Myc and
FLAG epitopes. Chemiluminescence (ECL, Amersham
Pharmacia Biotech, Amersham, Bucks, UK) was used to
detect the antibodies.
Enzymatic reactions and preparation of nucleotide
sugar samples
The yeast lysates with or without GMD a nd/or RMD were
incubated at 3 7 °C for 1 h in the presence of GDP-
D
-man-
nose, NADPH, NADP
+
,andMgCl
2
, after which 250 lL

of the reaction mixture was subjected to puri®cation before
HPLC analysis. Macromolecules were removed using
PD-10 columns (Amersham Pharmacia Biotech, Uppsala,
Sweden). The macromolecular fraction w as discarded, and
the micromolecular fraction was collected. After being dried
in a vacuum centrifuge and being redissolved, samples were
treatedfor30minat37°C with 50 U alkaline phosphatase
(Finnzymes, Espoo, Finland), which removed the phos-
phate groups from the nucleotides but left the nucleoside
diphosphate sugars intact. The reaction mixtures were
diluted with 10 m
M
NH
4
HCO
3
and applied to Bond Elut
columns (Varian, Harbor City, CA, USA) packed with
2 mL D EAE-Sepharose Fast Flow (Amersham Pharma-
cia). The anion-exchange columns were washed with 10 m
M
and 50 m
M
NH
4
HCO
3
, and the nucleotide sugars were then
eluted with 250 m
M

NH
4
HCO
3
.Afterbeingdriedand
redissolved in water several times, nucleotide sugars were
analyzed by HPLC.
HPLC methods
Nucleotide sugars were analyzed by ion-pair reversed-phase
HPLC on a Supelcosil LC-18 column (0.46 ´ 25 cm;
Supelco Inc., Bellafonte, PA, USA) a t a ¯ow rate of
1mLámin
)1
. Isocratic 10 m
M
triethylammonium acetate
buffer (pH 6.0) was used for 5 min, then a linear gradient of
0±3% acetonitrile in triethylammonium acetate buffer over
25 min. The ef¯uent was monitored with a UV detector at
254 nm.
Size-exclusion HPLC on a Superdex Peptide HR 10/30
column (Amersham Pharmacia Biotech) was performed at a
¯ow rate of 1 mLámin
)1
using 50 m
M
NH
4
HCO
3

,andthe
ef¯uent was monitored at 254 nm. The amount of GDP-
rhamnose in both HPLC methods was calculated from the
peak areas by reference to an external standard (GDP-
L
-fucose; Calbiochem). The samples containing GDP-
sugars were collected from HPLC runs for structural
analysis with MALDI-TOF MS and NMR.
Maldi-tof ms
MALDI-TOF MS was performed with a Bi¯ex mass
spectrometer (Bruker Daltonics, Leipzig, Germany).
Analysis was performed in the negative-ion linear delayed-
extraction mode, using 2,4,6-trihydroxyacetonephenone
(Fluka Chemica) as a matrix [15]. External calibration was
performed with this matrix dimer and sialyl Lewis X
b-methylglycoside (Toronto Research Chemicals, Toronto,
Ontario, Canada).
NMR experiments
An 11-nmol sample of GDP-rhamnose was dissolved in
300 lLD
2
O (Aldrich) and freeze-dried. The sample was
thendissolvedin40lLD
2
O. All NMR experiments w ere
carried out a t 35 °C on a 500-MHz Varian Inova
spectrometer equipped with a nanoprobe. The 1D
1
H-
NMR spectrum was recorded using a modi®cation of the

weft sequence for water suppression [16]. A total of 4096
transients were acquired with a spectral width of 6100 Hz.
For the DQF COSY spectrum [17], a total of 4000 ´ 256
complex data points were acquired, 256 transients per
increment. Before Fourier transformation, the data matrix
was multiplied by a cosine function in both dimensions. The
1
H chemical shifts were referenced to external 3-(trimethyl-
silyl)propionic-2,2,3,3-d4 acid (d  0).
Sequence analysis
The tools used for homology searches were mostly
BLAST
[18],
FASTA
[19], the Smith±Waterman implementation and
other programs available in the
GCG
package (Wisconsin
Package, version 10.0; Genetics Computer Group, Madison,
WI, USA). DNA sequences were aligned with the pro gram
PILEUP
(Wisconsin Package) or ClustalW (version 1.7) [20],
using an identity matrix, a gap weight of 8, and a gap length
weight of 0.1. Amino-acid sequences were aligned with the
same programs using a Blosum32 protein w eight matrix, a
gap weight of 12, and a gap length weight of 0.5. The DNA
alignments were checked by eye using the
GENEDOC
program
[21] and corrected to avoid alignments with disrupted

reading frames. Trees were constructed from the data using
maximum parsimony using programs from the
PHYLIP
[22]
package and the GCG implementation of
PAUP
* (Wisconsin
Package). H euristic se arches were utilized in parsimony
analyses because o f the great number of taxa examined.
Branch swapping was done by tree bisection±reconnection.
Ó FEBS 2002 Biosynthesis of GDP-rhamnose (Eur. J. Biochem. 269) 595
Bootstrap analyses (unshown) of 1000 replicates were
performed to examine the relative support of each relation-
ship in the resultant topologies. GeneDoc an d TreeView [23]
were used to prepare illustrations of the alignments and
the trees.
RESULTS
Cloning of
P. aeruginosa rmd
into a
S. cerevisiae
expression vector
We have previously cloned the gmd and wcaG genes of
E. coli and the gmd and wbcJ genes of H. pylori into pESC-
LEU, a yeast expression vector with two separate multiple
cloning sites. With these double constructs, we generated
S. ce revisiae strains that converted yeast endogenous GDP-
D
-mannose into GDP-
L

-fucose ([7]; N. Ja
È
rvinen,M.Ma
È
ki,
J. Ra
È
bina
È
, C. Roos, P. Mattila & R. Renkonen, unpub-
lished). In the present study, we aimed to produce GDP-
rhamnose from the 4-keto-6-deoxy intermediate synthesized
by the H. pylori GMD enzyme, and t herefore we identi®ed,
cloned, and expressed the P. aeruginosa rmd gene together
with the H. pylori gmd gene.
TheA-bandgeneclusterofP. aeruginosa (EMBL/
GenBank/DDBJ accession number AE004958) containing
the putative rmd gene was obtained from the database. The
size of the putative rmd gene was 915 bp, and the starting
codon was TTG, not ATG as suggested by the study of
Rocchetta et al.[9].Thermd gene was ampli®ed from
P. aeruginosa ATCC 27853 chromosomal DNA and cloned
into the expression vectors, pESC-LEU and pHP1 in-frame
with an N-terminal FLAG epitope , yielding pRHA1 and
pRHA2, respectively (Fig. 2). The sequenced plasmids
pHP1, pRHA1 and pRHA2 were subsequently transformed
into the expression strain S. ce revisiae YPH501.
Expression of GMD and RMD proteins in
S. cerevisiae
The H. pylori gmd and P. aeruginosa rmd genes were

expressed under g alactose-inducible promoters of the
pESC-LEU vector tagged with the c-Myc and FLAG
epitopes, respectively (Fig. 2). After induction, expression of
GMD and RMD proteins was analyzed in the yeast lysates
by Western immunoblots using antibodies against the
c-Myc and FLAG epitopes. As shown in Fig. 3, the presence
of the 44-kDa GMD could be detected in the lysates of
S. ce revisiae YPH501(pHP1) and YPH501(pRHA2). The
de no vo expressed FLAG-tagged putative RMD protein was
present in the cell lysates of YPH501(pRHA1) and
YPH501(pRHA2) (Fig. 3). The size o f this protein wa s
34 k Da, which corresponded to the calculated molecular
mass of RMD (33.9 kDa). No relevant bands could be
detected from the lysate of the YPH501(pESC-LEU) strain
used as a vector control (Fig. 3).
Characterization of enzymatic activities
To show that GMD and RMD were functionally active, we
performed a thorough analysis of sugar nucleotides formed
in reactions of yeast lysates with exogenously added GDP-
D
-mannose. The s ugar nucleotides from yeast lysates were
analyzed by HPLC, MALDI-TOF MS and
1
HNMR.
The ion-pair reversed-phase HPLC analysis (Fig. 4 )
showed that the vector control S. cerev isiae YPH501
(pESC-LEU) gave only a peak with the same retention
time as the GDP-
D
-mannose standard at 17.4 min and some

uncharacterized peaks from yeast cells (Fig. 4A). The peaks
from the yeast strain YPH501(pRHA1) expressin g RMD
were similar to the vector control (Fig. 4B). In contrast,
YPH501(pHP1) expressing GMD gave a smaller GDP-
D
-mannose peak and a novel peak at 21.2 min (Fig. 4C). As
no standard was a vailable for this intermediate product, we
isolated it from HPLC and tried to analyze its mass by
MALDI-TOF MS. However, we could not analyze it with
con®dence, possibly b ecause 4-keto-6-deoxysugars are
known to be labile. The presence of the 4-keto-6-deoxy
intermediate in the reaction mixture was also studied by
chemical reduction with NaBH
4
. As expected, two new
peaks were detected by HPLC (not shown). The minor peak
co-migrated with the GDP-
D
-rhamnose standard, and the
retention time of the major peak was near that of the 4-keto
intermediate product. The latter is probably GDP-deoxy-
D
-talose, but we were not able to con®rm this because of the
lack of a standard for this nucleotide sugar.
Fig. 2. Schematic drawing of the pRHA2 plasmid. The pRHA2 plas-
mid is a derivative of the yeast expression vector, pESC-LEU. The
H. pylori gmd gene was s ubclon ed under the GAL1 promoter in-frame
with the c-Myc epitope, and the P. aeruginosa rmd ge ne was subclon ed
under the GAL10 promoter in-frame with the FLAG epitope.
Fig. 3. Western blots of the expression of GMD and RMD in S. cere-

visiae YPH501 detected with c-Myc antibody (A) and FLAG antibody
(B). Lane 1, YPH501(pESC-LEU), the vector control; lane 2,
YPH501(pHP1); lane 3, YPH501(pRHA1); lane 4, YPH501(pRHA2).
The pre sence of 44-kDa H. pylori GMD was detected in lanes 2 and 4,
and the presence of 34-kD a P. aeruginosa RMD in lanes 3 and 4.
596 M. Ma
È
ki et al.(Eur. J. Biochem. 269) Ó FEBS 2002
In HPLC analysis of the double-construct strain
YPH501(pRHA2) expressing both GMD and RMD, the
4-keto-6-deoxy intermediate was not detected, but a novel
peak appeared at 19.2 min (Fig. 4D). Once again, there was
no commercially available s tandard for GDP-
D
-rhamnose,
but we could show that the molecule with a retention time of
19.2 min gave a single peak at m/z 588.05 on MALDI-TOF
MS analysis, which is the mass of GDP-deoxyhexoses
(calculated m/z for [ M-H]
±
is 588.08). This new GDP-
deoxyhexose peak, together with the putative 4-keto-
6-deoxy intermediate peak, was also seen when the lysates
of YPH501(pHP1), expressing GMD, and YPH501
(pRHA1), expressing RMD, were mixed together (Fig. 4E).
Formation of the putative GDP-rhamnose product was
further increased in this reaction, as compared with the
double-construct strain YPH501(pRHA2). As the retention
time of the reaction product was different from the GDP-
L

-fucose standard in ion-pair reversed-phase HPLC (19.2
and 21.6 min, respectively), it clearly represented a novel
GDP-deoxyhexose, and we therefore analyzed it further by
NMR (see below).
Preparative synthesis and puri®cation of GDP-rhamnose
The ®nal reaction product was puri®ed from the reaction
mixture to con®rm its structure and con®guration. In the
large-scale puri®cation of GDP-rhamnose for NMR ana-
lysis, new sample preparation techniques developed in our
laboratory for nucleotide sugars were used (J. Ra
È
bina
È
,M.
Ma
È
ki,N.Ja
È
rvinen, E. Saulahti & R. Renkonen, unpub-
lished results). Sh ortly, after the puri®cation on Envi-Carb
graphite columns (Supelco), DEAE-Sepharose anion-
exchange chromatography and r eversed-phase HPLC iden-
tical with the analytical runs (see above) were performed.
After further puri®cation by size-exclusion HPLC,
 11 nmol sugar nucleotide was pooled from several
HPLC runs.
The yield of GDP-rhamnose after the puri®cation steps
was determined from HPLC of the product (see Experi-
mental procedures). As calculated from the GDP-
D

-man-
nose added t o the cell e xtracts, 3±4% of the substrate was
converted into GDP-rhamnose in the independent experi-
ments with the double-construct strain, S. cerevisiae
YPH501(pRHA2). When the yeast lysates of strains
YPH501(pHP1), expressing GMD, and YPH501(pRHA1),
expressing RMD, were mixed and incubated together with
GDP-
D
-mannose, the yield of GDP-rhamnose was 9%.
NMR analysis
The
1
H-NMR spectrum (Fig. 5) of the puri®ed 19.2 min
peak from the HPLC pro®le (Fig. 4) was assigned, and the
proton±proton coupling constants
3
J
H,H
(Table 2) were
determined from a DQF COSY spectrum (not shown). The
NMR results established the structure as GDP-rhamnose,
propably the
D
-isomer. The coupling constants between the
ring protons of the rhamnosyl unit were characteristic of a
manno- con®guration and clearly distinguished the struc-
ture from GDP-
L
-fucose. The H6 signal at 1.270 p.p.m. was

on the region of a methyl group, and had the intensity of
three protons. T he large g eminal coupling t ypical of a
hydroxymethyl group was not observed. The chemical shifts
obtained were similar to those published by Kneidinger
et al. [8] for GDP-
D
-rhamnose and different from those
reported for GDP-
L
-fucose [24].
Conservation of RMD protein homologs among
different bacteria
After the enzymatic function had been con®rmed, th e RMD
protein of P. aeruginosa was u sed as a probe to ®nd more
putative RMD sequences from the databases based on the
primary sequence similarity. Relatively high homologies
were found with the Aneurinibacillus thermoaerophilus
RMD sequence (EMBL/GenBank/DDBJ accession num-
ber AF317224) as well as with three other bacterial ORFs of
Thiobacillus ferrooxidans (the TIGR accession number
gnl|TIGR|t_ferrooxidans_6147), Mycobacterium tuberculo-
sis (EMBL/GenBank/DDBJ accession number AL123456)
and Xylella fastidiosa (EMBL/GenBank/DDBJ accession
number AE003849). Signi®cant similarities were also found
with GDP-mannose dehydratase (EC 4.2.1.47), dTDP-
glucose dehydratase (EC 4.2.1.46) and UDP-glucose epi-
merase (EC 5.1.3.2) protein families. Therefore, we aligned
the selected gene sequences from the four enzyme families t o
evaluate the distance inter se. As a measure of distance w e
used the mutation rate, and, to visualize the result, we used

standard phylogenetic tools. The analysis showed that,
while the different genes clustered according to their
Fig. 4. HPLC analysis of the products of enzymatic reactions catalyzed
by H. pylori GMD and P. aeruginosa RMD. GDP-
D
-mannose was
incubated with t he lysates o f S. cere visiae YPH501 recombinant
strains. (A) YPH501(pESC-LEU), the vector control; (B) YPH501
(pRHA1) e xpressing RMD ; (C) YPH501(pHP1) expressing
GMD; (D) YHP501(pRHA2) coexpressing GMD and RMD;
(E) YPH501(pHP1) and YPH501(pRHA1), mixture of the lysates of
singularly expressed GMD and RMD. Peaks: M  GDP-
D
-man-
nose; R  GDP-rhamnose; K  GDP-4-keto-6-deoxy-
D
-mannose.
Ó FEBS 2002 Biosynthesis of GDP-rhamnose (Eur. J. Biochem. 269) 597
proposed function (Fig. 6), their relatedness within the
group was not much higher than between the groups. This
was further analyzed by aligning the sequences from the
GDP-mannose reductase group (EC 1.1.1.187): the seq-
uences of P. aeruginosa and A. thermoaerophilus with
proven RMD activities, and the ORFs of T. ferrooxidans,
M. tuberculosis and X. fastidiosa with putative RMD
activity. As can be deduced from the alignment (Fig. 7),
the sequences did not have any major stretches of similarity,
but rath er short patterns, most of which are also found in
the other three enzyme families (unshown).
The ®rst draft of the human genome (http://www.

celera.com) was also probed with the P. aeruginosa RMD
sequence, but no RMD analogue was found.
DISCUSSION
In this paper, we describe the molecular identi®cation of the
P. aeruginosa rmd gene and the enzymatic characterization
of the corresponding recombinant enzyme expressed in
S. ce revisiae YPH501. Using the yeast expression system,
we have had previous success in converting yeast endoge-
nous GDP-
D
-mannose into GDP-
L
-fucose by functionally
active E. coli GMD and GMER enzymes [10]. Now, we
also have a yeast expression system for synthesizing GDP-
rhamnose. Our results indicate that the yeast lysate can
convert exogenously added GDP-
D
-mannose into GDP-
rhamnose when P. aeruginosa RMD is e xpressed together
with H. pylori GMD. Because the level of yeast endogenous
GDP-
D
-mannose is probably not high enough for abundant
GDP-rhamnose production, we added exogenous GDP-
D
-mannose to the reaction mixture. It is likely that there are
Fig. 5. Structure of GDP-
D
-rhamnose (A) and

expansion and assignments of 500-MHz
1
H-NMR spectrum of GDP-rhamnose at 35 °C
(B). The signals arising from a small fraction
of impurities present in the sample are marked
with asterisks. In addition to the signals shown
in this expansion , H8 resonance of the guanine
unit was observed at 8.123 p.p.m.
Table 2 .
1
H chemical s hifts and coupling constants of GDP-
a-
D
-rhamnose. Chemical shifts were measured at 35 °C with reference
to external 3-(trimethylsilyl)propionic-2,2,3,3,-d4 a cid.
3
J
H, H+1
val-
ues from ®rst-order analysis of the DQF COSY spectrum. ND, Not
determined.
Residue Proton
Chemical
shift ( p.p.m.)
3
J
H, H+1
(Hz)
3
J

H,P
(Hz)
Rhamnose 1 5.446 < 2 6
2 4.051 2.6 ±
3 3.886 9.3 ±
4 3.435 10.0 ±
5 3.923 6.3 ±
6 1.270 ± ±
Ribose 1 5.942 ND ND
2 4.808 ND ±
3 4.529 ND ±
4 4.359 ND ±
5, 5¢ 4.22 ND ±
Guanine 8 8.123 ± ±
598 M. Ma
È
ki et al.(Eur. J. Biochem. 269) Ó FEBS 2002
enzymes other than H. pylori GMD in the crude yeast
lysate, such as mannosyltransferases [25,26], which also
compete for GDP-
D
-mannose. From the HPLC pro®les, we
calculated that most of the exogenously added GDP-
D
-mannose is converted into something other than GDP-
rhamnose (data not shown). However, puri®cation of the
GMD and RMD e nzymes and optimization of the reaction
conditions would probably lead to increased GDP-rham-
nose yield.
The biosynthesis of GDP-

D
-rhamnose, which acts as a
nucleotide sugar donor for
D
-rhamnosylation, received
increased interest after
D
-rhamnose was shown to b e an
essential extracellular and cell wall component of several
pathogenic bacteria [27]. P. aeruginosa is commonly isolated
Fig. 6. Grouping of four enzyme families UDP-glucose-4-epimerases
a
, dDTP-glucose-4,6-dehydratases
b
, GDP-4-keto-6-deoxy-
D
-mannose reductases
c
and GDP-mannose-4,6-dehydratases
d
on the basis of their sequence similarity. The scale bar indicates a ÔdistanceÕ in numb er of mutations per site and
the asterisks indicate the functionally characterized enzymes. EMBL/GenBank/DDBJ and TIGR accession numbers of the used gene sequences:
M. tuberculosis H37Rv (Z95436
a
, Z95390
b
, AL123456
c
, AL021926
d

); P. aeruginosa PAO1 (gnl|TIGR|PAGP_287
a
, AE004929
b
, AE004958
c
,
U18320
d
); E. coli K12 (X06226
a
, AE000294
b
, U38473
d
); X. fastidiosa 9a5c (AE003906
a,c
, AE003849
d
); A. thermoaerophilus L420-91
T
(AF317224
c
,
AF317224
d
); T. ferrooxidans ATCC 23270 (gnl|TIGR|t_ferrooxidans_6147
c
); S. enterica serovar typhimurium (X56793
b

); H. pylori J99
(AE001443
d
).
Fig. 7. Alignment of the known and putative RMDs from P. aeruginosa, A. thermoaerophilus, T. ferrooxidans, M. tu bercul osis and X. fastidiosa. The
alignment emphasizes the c onserved motifs, and the parentheses mark those conserved in the three other enzyme families (Fig. 6) as we ll.
Ó FEBS 2002 Biosynthesis of GDP-rhamnose (Eur. J. Biochem. 269) 599
from specimens obtained from lungs of patients with cystic
®brosis, and the respiratory P. aeruginosa isolates express
mainly
D
-rhamnosylated A-band lipopolysaccharide [1].
Interestingly, the same O-polysaccharide structure has
been isolated from the other opportunistic patho gens,
Burkholderia cepacia and Stenotrophomonas maltophilia,
that are also linked to this congenital monogenic disease
with severe pulmonary manifestations [28±30].
Research groups studying the relevance of rhamnosyla-
tion of various bacteria would bene®t from availability of
the building blocks required for synthesis of rhamnosylated
molecules. However, before these molecules can be synthe-
sized in vitro, the activated sugar nucleotides, GDP-
D
-rhamnose and dTDP-
L
-rhamnose, and the corresponding
rhamnosyltransferases that catalyze the speci®c glycosidic
linkages are needed.
Two enzymes responsible for RMD activity have recently
been characterized from the nonpathogenic bacterium

A. thermoaerophilus [8]. A. thermoaerophilus is a Gram-
positive bacterium, and the
D
-rhamnose residues are found
in the extracellular S-layer. A. thermoaerophilus GMD has
been proposed to be bifunctional, acting as a GDP-
mannose-4,6-dehydratase and a reductase. The latter activ-
ity was relatively weak compared with A. thermoaerophilus
RMD acting only as a reductase. In our analysis, we could
not show the bifunctionality of the H. pylori GMD enzyme,
which suggests that the speci®city of GMD enzymes varies
between bacterial species.
Currently, very little is known about bacterial rhamno-
syltransf era ses.
L
-Rhamnosyltransferases, which use dTDP-
L
-rhamnose as a donor, have been reported in several
bacteria [31±33], whereas putative
D
-rhamnosyltransferases,
which use GDP-
D
-rhamnose as a donor, have only been
identi®ed in P. aeruginosa [1] . It has been shown by muta-
genesis studies that these three putative
D
-rhamnosyltransfe-
rases participate in the synthesis of the A-band O-antigen.
If rhamnosylation is con®rmed to be essential for the

viability or virulence of pathogenic bacteria, the enzymes
involved in the biosynthesis of rhamnosylated glycans could
be ideal targets for antibacterial chemotherapy. Human
patients lack rhamnosylation and thus would probably not
suffer if enzymes involved in rhamnosylation were inhibited.
ACKNOWLEDGEMENTS
The work was supported in part by Research Grants f rom the Academy
of Finland,Technology Development Centre (TEKES), Helsinki, and
the Sigrid Juselius F oundation and a grant from the Helsinki University
Central Hospital Fund . We thank Dr Jari Helin and Leena Penttila
È
for
the MALDI-TOF M S analysis. Sirkka-Liisa Kaur anen and T uula
Kallioinen are thanked for skilled technical assistance with the
molecular biology, and Jonna-Mari Ma
È
ki for invaluable help with
the ®gures.
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