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

269, 593-601 2002 © FEBS 2002 Functional expression of Pseudomonas aeruginosa GDP-4-keto- 6-deoxy-p-mannose reductase which synthesizes GDP-rhamnose Minna Maki’, Nina Jarvinen’, Jarkko

Eur J Biochem 269, 593-601 (2002) © FEBS 2002

Functional expression of Pseudomonas aeruginosa GDP-4-keto-

6-deoxy-p-mannose reductase which synthesizes GDP-rhamnose

Minna Maki’, Nina Jarvinen’, Jarkko Rabina', Christophe Roos”, Hannu Maaheimo’,

Pirkko Mattila? and Risto Renkonen’

‘Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, University of Helsinki, Finland;

> MediCel, Helsinki, Finland; 3VTT Biotechnology, Espoo, Finland; 4HUCH Laboratory Diagnostics,

Helsinki University Central Hospital, Helsinki, Finland

Pseudomonas aeruginosa is an opportunistic Gram-negative

bacterium that causes 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-p-rhamnose, the p-rhamnose donor in

b-rhamnosylation, starts from GDP-p-mannose It is first

converted by the GDP-mannose-4,6-dehydratase (GMD)

into GDP-4-keto-6-deoxy-p-mannose, and then reduced to

GDP-p-rhamnose by GDP-4-keto-6-deoxy-p-mannose

reductase (RMD) Here, we describe the enzymatic char-

acterization of P aeruginosa RMD expressed in Sacchar-

omyces cerevisiae Previous success in functional expression

of bacterial gid genes in S cerevisiae allowed us to convert

GDP-p-mannose into GDP-4-keto-6-deoxy-p-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 confirmed to be GDP- rhamnose by HPLC, matrix-assisted laser desorption/ion- ization time-of-flight MS, and finally NMR spectroscopy The functional expression of P aeruginosa RMD in

S cerevisiae will provide a tool for generating GDP-rham- nose for in vitro rhamnosylation of glycoprotein and glyco- peptides

Keywords: A-band O-antigen; GDP-4-keto-6-deoxy- bD-mannosereductase(RMD);GDP-rhamnose; Pseudomonas

aeruginosa

Pseudomonas aeruginosa is an opportunistic Gram-negative

bacterium that can cause infections in immunocompro-

mised patients including those with severe burn wounds,

cystic fibrosis and cancer The lipopolysaccharides that are

cell surface molecules and virulence factors 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

b-rhamnose sugar residues arranged as repeating trisaccha-

ride units (-3p-Rhaol—2p-Rhaw1—3p-Rhaa1-),, [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 p, the former is much more common The

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: Risto.Renkonen(@Helsinki.FI

Abbreviations GMD, GDP-mannose-4,6-dehydratase; RMD, GDP-

4-keto-6-deoxy-p-mannose reductase, MALDI-TOF, matrix-assisted

laser desorption/ionization time-of-flight

(Received 4 October 2001, accepted 19 November 2001)

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-p-rhamnose, the precursor of D-rhamnosylated glycans, was proposed in the 1960s [5] It

starts from GDP-p-mannose, which is first converted into

GDP-4-keto-6-deoxy-b-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-.-fucose, GDP-p-rhamnose, GDP-deoxy-p-talose

or GDP-deoxy-p-altrose, by separate enzymes (in the case

of GDP-.-fucose the 3,5-epimerization occurs before reduction) It is also an intermediate in the conversion of GDP-b-mannose into the GDP-dideoxyhexose, GDP-col- itose [6], and the GDP-dideoxy amino sugar, GDP-p-per-

osamine [7] In the pathway of GDP-p-rhamnose synthesis,

the GDP-4-keto-6-deoxy-b-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 ef 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-p-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 gene and to synthesize 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

GDP-D-mannose

GDP-mannose

4,6 dehydratase

(GMD)

GDP-4-keto-6-deoxy-D-mannose

4-reductase 3-epimerase

4-reductase

3, 5-epimerase 4-reductase

4-reductase (RMD)

(GMER)

GDP-L-fucose GDP-D-rhamnose GDP-6-deoxy- GDP-6-deoxy-

Fig 1 Biosynthetic pathways of the deoxyhexoses from the common

4-keto-6-deoxy intermediate GDP-p-mannose is first converted into

the 4-keto-6-deoxy intermediate, which is then reduced to different

deoxyhexoses The enzymes involved in the GDP-p-fucose and GDP-

D-rhamnose pathways have been characterized, whereas the reaction

steps and enzymes involved in the GDP-deoxy-p-talose and GDP-

deoxy-b-altrose pathways have not been identified

deoxyhexose sugar nucleotides [10] because the glycosyla-

tion is largely restricted to mannosylation and yeast is not

known to have deoxyhexose metabolism of its own [11,12]

Therefore, the cytoplasm of yeast cells 1s a relatively rich

source of GDP-p-mannose, the starting material for the

rhamnose pathway, without any competing endogenous

enzymatic activity As detection and identification of

various deoxyhexoses is not straightforward, the lysates of

yeast transformants were analyzed by HPLC, matrix-

assisted laser desorption/ionization time-of-flight (MAL-

DI-TOF) MS and 'H NMR to verify the structure of the

synthesized GDP-rhamnose

Table 1 Bacterial and yeast strains and plasmids

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 the 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 pgmL' kanamycin and 100 pgmL~' ampicillin

Recombinant DNA techniques Chromosomal DNA was isolated from P aeruginosa ATCC 27853 using a QJAamp Tissue kit (Qiagen, Hilden, Germany) The rmd gene was amplified from chromosomal DNA using the primer set RMDF 5’-GAAGATCTTA ACTCAGCGTCTGTTCGTC (creating a Bg/II site) and RMDR_ 5’-GGTTAATTAATCAGATAAAAGGCCCG CTT (creating a Pacl site) The PCR product was first 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 Bg/II/Pacl sites

of the pESC-LEU (Stratagene, La Jolla, CA, USA) and

the pHPI (N Jarvinen, M Maki, J Rabina, C Roos,

P Mattila & R Renkonen, unpublished) vectors in-frame with an N-terminal FLAG epitope, yielding the corre- sponding plasmids pRHAI and pRHA2 pHPI! 1s a derivative of the pESC-LEU vector containing the H pylori gmd gene cloned in-frame with an N-terminal c-Myc epitope

Strain

P aeruginosa

E coli

AlacX74 deoR recA1 aral139 A(ara-leu)7697 galU galK rpsL(Str®) endA1 nupG

S cerevisiae

YPHS01 ura3—52 Iys2—8012PET „jJ¿2—1019°hfÊ rrp1-A63 Stratagene

his3-A200 feu2-Al, mating type a/o Plasmid

gmd gene as a 1143-bp fragment under GAL10 promoter et al (unpublished)

rmd gene as a 915-bp fragment under GAL] promoter

under GAL10 promoter and the P aeruginosa rmd gene under GALI] promoter

© FEBS 2002

(N Jarvinen, M Maki, J Rabind, C Roos, P Mattila &

R Renkonen, unpublished) The recombinant plasmids

were sequenced on an automated ABI 3100 sequencer (PE

Biosystems) pESC-LEU, pHP1l, pRHAI 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 GALI and GALI10 promoters of the pESC-LEU

expression vector are repressed by dextrose and induced by

galactose In the expression experiments, the yeast strains

were first 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 30 °C The yeast cells were

harvested and lysed with Y-PER Yeast Protein Extraction

reagent (2.5 mL for 1 g cell paste; Pierce, Rockford, IL,

USA) supplemented with 5mm MgCl, 200 um GDP-

b-mannose (Sigma, St Louis, MO, USA), 200 tw NADP *

(Calbiochem, San Diego, CA, USA) and 200 um 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 using 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 and/or RMD were

incubated at 37 °C for 1 h in the presence of GDP-p-man-

nose, NADPH, NADP’, and MgCh, after which 250 HL

of the reaction mixture was subjected to purification before

HPLC analysis Macromolecules were removed using

PD-10 columns (Amersham Pharmacia Biotech, Uppsala,

Sweden) The macromolecular fraction was discarded, and

the micromolecular fraction was collected After being dried

in a vacuum centrifuge and being redissolved, samples were

treated for 30 min at 37 °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 mm NH,HCO; and applied to Bond Elut

columns (Varian, Harbor City, CA, USA) packed with

2 mL DEAE-Sepharose Fast Flow (Amersham Pharma-

cia) The anion-exchange columns were washed with 10 mm

and 50 mm NH4HCOs, and the nucleotide sugars were then

eluted with 250 mm NH,HCO3 After being dried and

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 x 25 cm;

Supelco Inc., Bellafonte, PA, USA) at a flow rate of

Biosynthesis of GDP-rhamnose (Eur J Biochem 269) 595

1 mL-min“' Isocratic 10 mm 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 effluent 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

flow rate of 1 mL-min™! using 50 mm NH,HCOs,, and the

effluent 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 Biflex 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 1|1l-nmol sample of GDP-rhamnose was dissolved in

300 nL DO (Aldrich) and freeze-dried The sample was then dissolved in 40 nL DO All NMR experiments were carried out at 35°C on a 500-MHz Varian Inova spectrometer equipped with a nanoprobe The 1D 'H- NMR spectrum was recorded using a modification 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 x 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 'H chemical shifts were referenced to external 3-(trimethyl- silyl)propionic-2,2,3,3-d4 acid (6 = 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 program 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 weight 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) Heuristic searches were utilized in parsimony analyses because of the great number of taxa examined Branch swapping was done by tree bisection—reconnection

Bootstrap analyses (unshown) of 1000 replicates were

performed to examine the relative support of each relation-

ship in the resultant topologies GeneDoc and 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 coliand the gmd and whcJ genes of H pylori into pESC-

LEU, a yeast expression vector with two separate multiple

cloning sites With these double constructs, we generated

S cerevisiae strains that converted yeast endogenous GDP-

D-mannose into GDP-L-fucose ([7]; N Jarvinen, M Maki,

J Rabind, 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 therefore we identified,

cloned, and expressed the P aeruginosa rmd gene together

with the H pylori gmd gene

The A-band gene cluster of P 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] The rmd gene was amplified from

P aeruginosa ATCC 27853 chromosomal DNA and cloned

into the expression vectors, pPESC-LEU and pHP1 in-frame

with an N-terminal FLAG epitope, yielding pRHAI and

pRHA2, respectively (Fig 2) The sequenced plasmids

pHP1, pRHAI and pRHA2 were subsequently transformed

into the expression strain S cerevisiae YPH501

Expression of GMD and RMD proteins in S cerevisiae

The H pylori gmd and P aeruginosa rmd genes were

expressed under galactose-inducible promoters of the

LEU2

rmd

pRHA2 oe

PGALI C-MYC gmd

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 subcloned under the GAL1 promoter in-frame

with the c-Myc epitope, and the P aeruginosa rmd gene was subcloned

under the GAL10 promoter in-frame with the FLAG epitope

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 cerevisiae YPHS01(pHP1) and YPH501(pRHA2) The

de novo expressed FLAG-tagged putative RMD protein was present in the cell lysates of YPHSOI(pRHAI) and YPHS01(pRHA2) (Fig 3) The size of this protein was

34 kDa, 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-LEV) 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 sugar nucleotides from yeast lysates were analyzed by HPLC, MALDI-TOF MS and 'H NMR The ion-pair reversed-phase HPLC analysis (Fig 4) showed that the vector control S cerevisiae YPH501 (pESC-LEU) gave only a peak with the same retention time as the GDP-p-mannose standard at 17.4 min and some uncharacterized peaks from yeast cells (Fig 4A) The peaks from the yeast strain YPH501(pRHA1) expressing RMD were similar to the vector control (Fig 4B) In contrast, YPHS501(pHP1) expressing GMD gave a smaller GDP- D-mannose peak and a novel peak at 21.2 min (Fig 4C) As

no standard was available 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 confidence, possibly because 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 NaBHy As expected, two new peaks were detected by HPLC (not shown) The minor peak co-migrated with the GDP-p-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-

p-talose, but we were not able to confirm this because of the

lack of a standard for this nucleotide sugar

—

Fig 3 Western blots of the expression of GMD and RMD in S cere- visiae YPH4501 detected with c-Myc antibody (A) and FLAG antibody (B) Lane 1, YPHSO01(pESC-LEU), the vector control; lane 2, YPHS01(pHP1); lane 3, YPH501(pRHAL); lane 4, YPH501(pRHA2) The presence of 44-kDa H pylori GMD was detected in lanes 2 and 4, and the presence of 34-kDa P aeruginosa RMD in lanes 3 and 4.

© FEBS 2002

M

R

Time (min)

Fig 4 HPLC analysis of the products of enzymatic reactions catalyzed

by H pylori GMD and P aeruginosa RMD GDP-p-mannose was

incubated with the lysates of S cerevisiae YPHS0O1 recombinant

strains (A) YPHSOI(pESC-LEU), the vector control; (B) YPH501

(pRHAI) expressing RMD; (C) YPHSOI(pHP1)_ expressing

GMD; (D) YHPS0l(pRHA2) coexpressing GMD and RMD;

(E) YPHS01(pHP1) and YPH501(pRHA1), mixture of the lysates of

singularly expressed GMD and RMD Peaks: M = GDP-p-man-

nose; R = GDP-rhamnose; K = GDP-4keto-6-deoxy-pb-mannose

In HPLC analysis of the double-construct strain

YPHS01(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 standard for GDP-p-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] 1s 588.08) This new GDP-

deoxyhexose peak, together with the putative 4keto-

6-deoxy intermediate peak, was also seen when the lysates

of YPH50I(pHPI), expressing GMD, and YPH250I

(pDRHA1), 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 10n-pair reversed-phase HPLC (19.2

and 21.6 min, respectively), it clearly represented a novel

GDP-deoxyhexose, and we therefore analyzed 1t further by

NMR (see below)

Preparative synthesis and purification of GDP-rhamnose

The final reaction product was purified from the reaction

mixture to confirm its structure and configuration In the

Biosynthesis of GDP-rhamnose (Eur J Biochem 269) 597

large-scale purification of GDP-rhamnose for NMR ana- lysis, new sample preparation techniques developed in our laboratory for nucleotide sugars were used (J Rabina, M

Maki, N Jarvinen, E Saulahti & R Renkonen, unpub-

lished results) Shortly, after the purification on Envi-Carb graphite columns (Supelco), DJEAE-Sepharose anion- exchange chromatography and reversed-phase HPLC iden- tical with the analytical runs (see above) were performed After further purification by size-exclusion HPLC,

~11 nmol sugar nucleotide was pooled from several HPLC runs

The yield of GDP-rhamnose after the purification steps was determined from HPLC of the product (see Experi- mental procedures) As calculated from the GDP-p-man-

nose added to the cell extracts, 3-4% of the substrate was

converted into GDP-rhamnose in the independent experi-

ments with the double-construct strain, S cerevisiae

YPHSOI(pRHA2) When the yeast lysates of strains YPHSOI(pHP1), expressing GMD, and YPHS501(pRHA1), expressing RMD, were mixed and incubated together with GDP-p-mannose, the yield of GDP-rhamnose was 9%

NMR analysis The 'H-NMR spectrum (Fig 5) of the purified 19.2 min peak from the HPLC profile (Fig 4) was assigned, and the proton-proton coupling constants JE (Table 2) were determined from a DQF COSY spectrum (not shown) The

NMR results established the structure as GDP-rhamnose,

propably the p-isomer The coupling constants between the ring protons of the rhamnosyl unit were characteristic of a manno- configuration and clearly distinguished the struc- ture from GDP-.-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 The large geminal coupling typical of a hydroxymethyl group was not observed The chemical shifts obtained were similar to those published by Kneidinger

et al [8] for GDP-p-rhamnose and different from those reported for GDP-.-fucose [24]

Conservation of RMD protein homologs among different bacteria

After the enzymatic function had been confirmed, the RMD protein of P aeruginosa was used as a probe to find 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 enl|TIGR|t_ ferrooxidans 6147), Mycobacterium tuberculo- sis (EMBL/GenBank/DDBJ accession number AL123456) and Xylella fastidiosa (EMBL/GenBank/DDBJ accession number AE003849) Significant 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 to evaluate the distance inter se As a measure of distance we

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

A

CH,

O

H

O O

HO O—-P-O-P-O-CH, _O R

H H OH OH

OH OH

B

Rib 5,5’

1

Rib Rib

Rha

Fig 5 Structure of GDP-p-rhamnose (A) and expansion and assignments of 500-MHz

"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

Table 2 'H chemical shifts and coupling constants of GDP-

o@-D-rhamnose Chemical shifts were measured at 35 °C with reference

to external 3-(trimethylsilyl)propionic-2,2,3,3,-d4 acid “Tu, H+1 Val-

ues from first-order analysis of the DQF COSY spectrum ND, Not

determined

Chemical Residue Proton — shift ( p.p.m.) Sg H+ 1 (Hz) Jp (Hz)

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

in this expansion, H8 resonance of the guanine unit was observed at 8.123 p.p.m

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 rather short patterns, most of which are also found in the other three enzyme families (unshown)

The first 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 identification of the

P aeruginosa rmd gene and the enzymatic characterization

of the corresponding recombinant enzyme expressed in

S cerevisiae YPHS01 Using the yeast expression system,

we have had previous success 1n converting yeast endoge- nous GDP-p-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-p-mannose into GDP- rhamnose when P aeruginosa RMD 1s expressed together with H pylori GMD Because the level of yeast endogenous GDP-p-mannose 1s 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

© FEBS 2002

UDP-glucose 4-epimerase

Mycobacterium tuberculosis

Pseudomonas aeruginosa

Escherichia coli *

Xylella fastidiosa

Aneurinibacillus thermoaerophilus *

Mycobacterium tuberculosis

Thiobacillus ferrooxidans

Pseudomonas aeruginosa *

Xylella fastidiosa 0.1

GDP-4-keto-6-deoxy-D-mannose reductase

Biosynthesis of GDP-rhamnose (Eur J Biochem 269) 599

dTDP-glucose 4,6-dehydratase

Mycobacterium tuberculosis

Pseudomonas aeruginosa

Salmonella enterica serovar typhimurium *

Escherichia coli *

Escherichia coli *

Helicobacter pylori *

Mycobacterium tuberculosis

Pseudomonas aeruginosa

Aneurinibacillus thermoaerophilus *

Xylelia fastidiosa

GDP-mannose 4,6-dehydratase

Fig 6 Grouping of four enzyme families UDP-glucose-4-epimerases*, dDTP-glucose-4,6-dehydratases”, GDP-4-keto-6-deoxy-p-mannose reductases‘ and GDP-mannose-4,6-dehydratases' on the basis of their sequence similarity The scale bar indicates a ‘distance’ in number 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*, Z95390°, AL123456°, AL021926°); P aeruginosa PAOL (gnl/TIGR|PAGP_287?, AE004929°, AE004958°,

U18320°); E coli K12 (X06226", AE000294°, U38473%); X fastidiosa 9a5c (AE003906"*, AE003849°); A thermoaerophilus L420-917 (AF317224°,

AF3I17224); T ferrooxidans ATCC 23270 (gnITIGRIt ferrooxidans 61479; S enterica serovar typhimurium (X56793°); H pylori J99

(AE001443°)

A thermoaerophilus 9 -

M tuberculosis

T ferrooxidans

P aeruginosa

X, fastidiosa

*

M tuberculosis PAVSy, ARPVEOLT R-VRPHAR: ` ae x7 TOMIAY SY,MH

T ferrooxidans PAA DE \PHESED UNGOS -SGEMGR MEVGRGD TZ SEAD P 2915“ FE

P aeruginosa Gig PEAGRUPARLLO -RGFSGTFEYISIGDVWEO AE SIỆC1,* a

X fastidiosa AQ JAHGSA,)~ ~DFY¥ THGDATPECTBLAS N -RERLP

A thermoaerophilus

M tuberculosis

ý N

T ferrooxidans TAG-VEATI—

P aeruginosa Saptiag- VEŸ7rIV

X fastidiosa CRDNTG- -HANAVET

Fig 7 Alignment of the known and putative RMDs from P aeruginosa, A thermoaerophilus, T ferrooxidans, M tuberculosis and X fastidiosa The alignment emphasizes the conserved motifs, and the parentheses mark those conserved in the three other enzyme families (Fig 6) as well

enzymes other than H pylori GMD in the crude yeast

lysate, such as mannosyltransferases [25,26], which also

compete for GDP-b-mannose From the HPLC profiles, we

calculated that most of the exogenously added GDP-

D-mannose is converted into something other than GDP-

rhamnose (data not shown) However, purification of the

GMD and RMD enzymes and optimization of the reaction

conditions would probably lead to increased GDP-rham- nose yield

The biosynthesis of GDP-p-rhamnose, which acts as a nucleotide sugar donor for pD-rhamnosylation, received increased interest after D-rhamnose was shown to be an essential extracellular and cell wall component of several pathogenic bacteria [27] P aeruginosa is commonly isolated

from specimens obtained from lungs of patients with cystic

fibrosis, and the respiratory P aeruginosa isolates express

mainly p-rhamnosylated A-band lipopolysaccharide [1]

Interestingly, the same O-polysaccharide structure has

been isolated from the other opportunistic pathogens,

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 benefit 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-

pb-rhamnose and dTDP-.-rhamnose, and the corresponding

rhamnosyltransferases that catalyze the specific 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 p-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 specificity of GMD enzymes varies

between bacterial species

Currently, very little is known about bacterial rhamno-

syltransferases L-Rhamnosyltransferases, which use dTDP-

L-rhamnose as a donor, have been reported in several

bacteria [31-33], whereas putative p-rhamnosyltransferases,

which use GDP-p-rhamnose as a donor, have only been

identified in P aeruginosa [1] It has been shown by muta-

genesis studies that these three putative p-rhamnosyltransfe-

rases participate in the synthesis of the A-band O-antigen

If rhamnosylation is confirmed 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 from the Academy

of Finland,Technology Development Centre (TEKES), Helsinki, and

the Sigrid Juselius Foundation and a grant from the Helsinki University

Central Hospital Fund We thank Dr Jari Helin and Leena Penttilé for

the MALDI-TOF MS analysis Sirkka-Liisa Kauranen and Tuula

Kallioinen are thanked for skilled technical assistance with the

molecular biology, and Jonna-Mari Maki for invaluable help with

the figures

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