Biochemical characterization of GDP-L-fucose de novo synthesis pathway in fungus Mortierella alpina

Journal Article · Fri Jan 22 04:00:00 EST 2010 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2009.12.116· OSTI ID:22202344

Ren, Yan ^[1]; Perepelov, Andrei V. ^[2]; Wang, Haiyan ^[1]; Zhang, Hao ^[3]; Knirel, Yuriy A. ^[2]; Wang, Lei ^[1]; Chen, Wei ^[3]

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1. TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China)
2. N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow (Russian Federation)
3. State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 (China)

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{sub 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 {sup o}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.

• OSTI ID: 22202344
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 4 Vol. 391; ISSN BBRCA9; ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
CAPILLARIES
CARBOXYLIC ACIDS
CATIONS
COENZYMES
CORRELATIONS
ELECTROPHORESIS
ENZYMES
ESCHERICHIA COLI
FUNGI
MANNOSE
MASS SPECTROSCOPY
NUCLEAR MAGNETIC RESONANCE
NUCLEOTIDES
PH VALUE
SACCHAROSE