Characterization of GDP-mannose dehydrogenase from the brown alga Ectocarpus siliculosus providing the precursor for the alginate polymer.

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.

Cloning of Glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars.

Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a K(m) of 0.7 mm. It requires ATP as phosphate donor (K(m) 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.

OCCURRENCE AND CHARACTERIZATION OF ARABINOGALACTAN-LIKE PROTEINS AND HEMICELLULOSES IN MICRASTERIAS (STREPTOPHYTA)(1).

The cell wall of the green alga Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zygnematophyceae, Streptophyta) was investigated to obtain information on the composition of component polysaccharides and proteoglycans to allow comparison with higher plants and to understand cell wall functions during development. Various epitopes currently assigned to arabinogalactan-proteins (AGPs) of higher plants could be detected in Micrasterias by immuno TEM and immunofluorescence methods, but the walls did not bind the ß-d-glycosyl-Yariv (ß-GlcY) reagent. Secretory vesicles and the primary wall were labeled by antibodies against AGPs (JIM8, JIM13, JIM14). Dot and Western blot experiments indicated a proteoglycan nature of the epitopes recognized, which consisted of galactose and xylose as major sugars by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Epitopes of alkali-soluble polysaccharides assigned to noncellulosic polysaccharides in higher plants could be detected and located in the wall during its formation. The polyclonal anti-xyloglucan (anti-XG) antibody labeled primary and secondary wall of Micrasterias, whereas the monoclonal antibody CCRC-M1, directed against the fucose/galactose side chain of xyloglucan (XyG), did not recognize any structures. Labeling by anti-XG antibody at the trans-sites of the dictyosomes and at wall material containing vesicles indicated that secretion of the epitopes occurred similar to higher plants. The presence of (1→3, 1→4)-ß-glucan (mixed linked glucan) in the secondary cell wall but not in the primary cell wall of Micrasterias could be demonstrated by an antibody recognizing this glucan type, whereas (1→3)-ß-glucan (callose) could not be detected. The analytical results revealed that alkali-soluble polysaccharides in the secondary wall of Micrasterias consist mostly of (1→3, 1→4)-ß-d-glucan.