Functional expression of Pseudomonas aeruginosa GDP-4-keto-6-deoxy-D-mannose reductase which synthesizes GDP-rhamnose

Minna Mäki, Nina Järvinen, Jarkko Räbinä, Christophe Roos, Hannu Maaheimo, Pirkko Mattila, Risto Renkonen (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

25 Citations (Scopus)

Abstract

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-d-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-d-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-d-mannose, and then reduced to GDP-d-rhamnose by GDP-4-keto-6-deoxy-d-mannose reductase (RMD). Here, we describe the enzymatic characterization of P.aeruginosa RMD expressed in Saccharomyces cerevisiae. Previous success in functional expression of bacterial gmd genes in S.cerevisiae allowed us to convert 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 confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization 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-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides.
Original languageEnglish
Pages (from-to)593-601
JournalEuropean Journal of Biochemistry
Volume269
Issue number2
DOIs
Publication statusPublished - 2002
MoE publication typeA1 Journal article-refereed

Fingerprint

Rhamnose
Guanosine Diphosphate Mannose
Mannose
Pseudomonas aeruginosa
Saccharomyces cerevisiae
Hydro-Lyases
Bacterial Genes
O Antigens
Genes
Glycopeptides
Gram-Negative Bacteria
Helicobacter pylori
Mammals
Lipopolysaccharides
Glycoproteins
Oxidoreductases
Lasers
Magnetic Resonance Spectroscopy
Yeast
High Pressure Liquid Chromatography

Keywords

  • gram-negative bacteria
  • Pseudomonas aeruginosa
  • infection
  • bacterial enzymes
  • enzymes

Cite this

Mäki, Minna ; Järvinen, Nina ; Räbinä, Jarkko ; Roos, Christophe ; Maaheimo, Hannu ; Mattila, Pirkko ; Renkonen, Risto. / Functional expression of Pseudomonas aeruginosa GDP-4-keto-6-deoxy-D-mannose reductase which synthesizes GDP-rhamnose. In: European Journal of Biochemistry. 2002 ; Vol. 269, No. 2. pp. 593-601.
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abstract = "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-d-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-d-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-d-mannose, and then reduced to GDP-d-rhamnose by GDP-4-keto-6-deoxy-d-mannose reductase (RMD). Here, we describe the enzymatic characterization of P.aeruginosa RMD expressed in Saccharomyces cerevisiae. Previous success in functional expression of bacterial gmd genes in S.cerevisiae allowed us to convert 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 confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization 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-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides.",
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author = "Minna M{\"a}ki and Nina J{\"a}rvinen and Jarkko R{\"a}bin{\"a} and Christophe Roos and Hannu Maaheimo and Pirkko Mattila and Risto Renkonen",
year = "2002",
doi = "10.1046/j.0014-2956.2001.02688.x",
language = "English",
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pages = "593--601",
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Functional expression of Pseudomonas aeruginosa GDP-4-keto-6-deoxy-D-mannose reductase which synthesizes GDP-rhamnose. / Mäki, Minna; Järvinen, Nina; Räbinä, Jarkko; Roos, Christophe; Maaheimo, Hannu; Mattila, Pirkko; Renkonen, Risto (Corresponding Author).

In: European Journal of Biochemistry, Vol. 269, No. 2, 2002, p. 593-601.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Functional expression of Pseudomonas aeruginosa GDP-4-keto-6-deoxy-D-mannose reductase which synthesizes GDP-rhamnose

AU - Mäki, Minna

AU - Järvinen, Nina

AU - Räbinä, Jarkko

AU - Roos, Christophe

AU - Maaheimo, Hannu

AU - Mattila, Pirkko

AU - Renkonen, Risto

PY - 2002

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N2 - 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-d-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-d-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-d-mannose, and then reduced to GDP-d-rhamnose by GDP-4-keto-6-deoxy-d-mannose reductase (RMD). Here, we describe the enzymatic characterization of P.aeruginosa RMD expressed in Saccharomyces cerevisiae. Previous success in functional expression of bacterial gmd genes in S.cerevisiae allowed us to convert 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 confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization 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-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides.

AB - 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-d-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-d-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-d-mannose, and then reduced to GDP-d-rhamnose by GDP-4-keto-6-deoxy-d-mannose reductase (RMD). Here, we describe the enzymatic characterization of P.aeruginosa RMD expressed in Saccharomyces cerevisiae. Previous success in functional expression of bacterial gmd genes in S.cerevisiae allowed us to convert 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 confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization 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-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides.

KW - gram-negative bacteria

KW - Pseudomonas aeruginosa

KW - infection

KW - bacterial enzymes

KW - enzymes

U2 - 10.1046/j.0014-2956.2001.02688.x

DO - 10.1046/j.0014-2956.2001.02688.x

M3 - Article

VL - 269

SP - 593

EP - 601

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 2

ER -