Abstract
Objective
To clarify the molecular mechanism of GDP-l-fucose biosynthesis in Mortierella alpina.
Results
Analysis of the M. alpina genome suggests that there were two isofunctional GDP-d-mannose-4,6-dehydratase genes (GMD1 and GMD2) that have never been found in a microorganism before. GMD2 was expressed heterologously in Escherichia coli and purified to homogeneity. The addition of exogenous NAD+ or NADP+ was not essential for GMD2 activity. GMD2 may have considerable importance for GDP-l-fucose biosynthesis under nitrogen starvation. The transcriptional regulation of GMD1 may be more susceptible to GDP and GTP than that of GMD2. Significant changes were observed in the concentration of GDP-l-fucose (30 and 36 % inhibition respectively) and total fatty acids (18 and 12 % inhibition respectively) in M. alpina grown on GMD inhibitors medium, which suggests that GDP-l-fucose is functionally significant in lipid metabolism.
Conclusions
This is the first time that an isofunctional GDP-d-mannose-4,6-dehydratase has been characterized in a microorganism.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Becker DJ, Lowe JB (2003) Fucose: biosynthesis and biological function in mammals. Glycobiology 13:41–53
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917
Bonin CP, Potter I, Vanzin GF, Reiter WD (1997) The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-d-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-l-fucose. Proc Natl Acad Sci 94:2085–2090
Chen H, Hao G, Wang L, Wang H, Gu Z, Liu L, Zhang H, Chen W, Chen YQ (2015) Identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi. Sci Rep 5:11247
Kendrick A, Ratledge C (1992) Lipids of selected molds grown for production of n-3 and n-6 polyunsaturated fatty acids. Lipids 27:15–20
Ma B, Simala-Grant JL, Taylor DE (2006) Fucosylation in prokaryotes and eukaryotes. Glycobiology 16:158–184
Mulichak AM, Bonin CP, Reiter WD, Garavito RM (2002) Structure of the MUR1 GDP-mannose 4,6-dehydratase from Arabidopsis thaliana: implications for ligand binding and specificity. Biochemistry 41:15578–15589
Ren Y, Perepelov AV, Wang H, Zhang H, Knirel YA, Wang L, Chen W (2010) Biochemical characterization of GDP-l-fucose de novo synthesis pathway in fungus Mortierella alpina. Biochem Biophys Res Commun 391:1663–1669
Sturla L, Bisso A, Zanardi D, Benatti U, De Flora A, Tonetti M (1997) Expression, purification and characterization of GDP-d-mannose 4,6-dehydratase from Escherichia coli. FEBS Lett 412:126–130
Sullivan FX, Kumar R, Kriz R, Stahl M, Xu GY, Rouse J, Chang XJ, Boodhoo A, Potvin B, Cumming DA (1998) Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro. J Biol Chem 273:8193–8202
Wang H, Yang B, Hao G, Feng Y, Chen H, Feng L, Zhao J, Zhang H, Chen YQ, Wang L, Chen W (2011a) Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina. Microbiology 157:3059–3070
Wang L, Chen W, Feng Y, Ren Y, Gu Z, Chen H, Wang H, Thomas MJ, Zhang B, Berquin IM, Li Y, Wu J, Zhang H, Song Y, Liu X, Norris JS, Wang S, Du P, Shen J, Wang N, Yang Y, Wang W, Feng L, Ratledge C, Chen YQ (2011b) Genome characterization of the oleaginous fungus Mortierella alpina. PLoS One 6:e28319
Wang H, Chen H, Hao G, Yang B, Feng Y, Wang Y, Feng L, Zhao J, Song Y, Zhang H, Chen YQ, Wang L, Chen W (2013) Role of the phenylalanine-hydroxylating system in aromatic substance degradation and lipid metabolism in the oleaginous fungus Mortierella alpina. Appl Environ Microbiol 79:3225–3233
Wang H, Zhang C, Chen H, Yang Q, Zhou X, Gu Z, Zhang H, Chen W, Chen YQ (2016) Characterization of a Fungal l-fucokinase involved in Mortierella alpina GDP-l-fucose salvage pathway. Glycobiology
Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. Biotechniques 39:75–85
Acknowledgments
This study was supported in part by the National Science Foundation of China (NSFC) (31400038, 31530056, and 31471128), the Program for New Century Excellent Talents (NCET-13-0831), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1249). This study is supported by program of “Collaborative innovation center of food safety and quality control in Jiangsu Province ”.
Supporting information
Supplementary Table 1—Gradient elution of the chromatographic separation.
Supplementary Table 2—Primers used.
Supplementary Table 3—Gene searching and transcriptome data analysis.
Supplementary Table 4—The GDP-L-fucose content and status of fatty acids in M. alpina grown on GMD inhibitors mediuma
Supplementary Figure 1—SDS-PAGE of GMD2.
Supplementary Figure 2—Multiple amino acid sequence alignment of GMD2 from different organisms.
Supplementary Figure 3—Phylogenetic tree of GMDs from different organisms.
Supplementary Figure 4—Liquid chromatography and mass spectrometry chromatographs.
Supplementary Figure 5—MS2 analysis of ion with m/z 586.06
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Wang, H., Zhang, C., Chen, H. et al. Biochemical characterization of an isoform of GDP-d-mannose-4,6-dehydratase from Mortierella alpina . Biotechnol Lett 38, 1761–1768 (2016). https://doi.org/10.1007/s10529-016-2153-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-016-2153-9