Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. L-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-L-fucose (the nucleotide-activated form of L-fucose) in M. alpina. Genes encoding GDP-D-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-L-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k(m) values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 degrees C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-L-fucose biosynthetic pathway in fungi. (C) 2009 Elsevier Inc. All rights reserved.