title: Characterization of GDP-mannose dehydrogenase from the brown alga Ectocarpus siliculosus providing the precursor for the alginate polymer.
abstract: Alginate is a major cell wall polymer of brown algae. The precursor for the polymer is GDP-mannuronic acid, which is believed to be derived from a four-electron oxidation of GDP-mannose through the enzyme GDP-mannose dehydrogenase (GMD). So far no eukaryotic GMD has been biochemically characterized. We have identified a candidate gene in the Ectocarpus siliculosus genome and expressed it as a recombinant protein in Escherichia coli. The GMD from Ectocarpus differs strongly from related enzymes in bacteria and is as distant to the bacterial proteins as it is to the group of UDP-glucose dehydrogenases. It lacks the C-terminal ∼120 amino acid domain present in bacterial GMDs, which is believed to be involved in catalysis. The GMD from brown algae is highly active at alkaline pH and contains a catalytic Cys residue, sensitive to heavy metals. The product GDP-mannuronic acid was analyzed by HPLC and mass spectroscopy. The K(m) for GDP-mannose was 95 μM, and 86 μM for NAD(+). No substrate other than GDP-mannose was oxidized by the enzyme. In gel filtration experiments the enzyme behaved as a dimer. The Ectocarpus GMD is stimulated by salts even at low molar concentrations as a possible adaptation to marine life. It is rapidly inactivated at temperatures above 30 °C.
authors: Department of Cell Biology, Division of Plant Physiology, University of Salzburg, 5020 Salzburg, Austria. raimund.tenhaken@sbg.ac.at
authors: Raimund
authors: R
authors: Tenhaken
authors: Elena
authors: E
authors: Voglas
authors: J Mark
authors: JM
authors: Cock
authors: Volker
authors: V
authors: Neu
authors: Christian G
authors: CG
authors: Huber
mesh_terms: D020816:Amino Acid Motifs; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry*; D002473:Cell Wall / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D009702:Nucleoside Diphosphate Sugars / Q000737:chemistry; D000459:Phaeophyceae / Q000201:enzymology*; D011108:Polymers / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D009702:Nucleoside Diphosphate Sugars; D011108:Polymers; C037228:GDP mannuronic acid; D006153:Guanosine Diphosphate; D002237:Carbohydrate Dehydrogenases; C020562:GDPmannose dehydrogenase