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

Pseudomonas aeruginosais an opportunistic Gram-negative bacterium that causes severe infections in a number of hosts from plants to mammals. A-band lipopolysaccharide of P. aeruginosacontains D-rhamnosylated O-antigen. The synthesis of GDP-D-rhamnose, the D-rhamnose donor in D-rhamnosylation, starts from GDP-D-mannose. | Eur. J. Biochem. 269 593-601 2002 FEBS 2002 Functional expression of Pseudomonas aeruginosa GDP-4-keto-6-deoxy-D-mannose reductase which synthesizes GDP-rhamnose Minna Maki1 Nina Jarvinen1 Jarkko Rabina1 Christophe Roos2 Hannu Maaheimo3 Pirkko Mattila2 and Risto Renkonen1 1 Department of Bacteriology and Immunology Haartman Institute and Biomedicum University of Helsinki Finland 2MediCel 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-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 Sacchar-omyces 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 confrmed to be GDP-rhamnose by HPLC matrix-assisted laser desorption ion-ization time-of-flight MS and fnally 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 glycopeptides. Keywords A-band O-antigen GDP-4-keto-6-deoxy-D-mannose reductase RMD GDP-rhamnose Pseudomonas aeruginosa. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that can cause infections in .