Abstract: Guanosine 5′-diphospho-β-L-fucose (GDP-L-Fuc) is a key intermediate for fucosylated oligosaccharides biosynthesis in plants and bacteria. In this study, a gene encoding L-fucose kinase/GDP-L-fucose pyrophosphorylase (FKP) from Bacteroides fragilis was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). The purified recombinant FKP had the maximum activity at 45 ℃ and 7.5. The enzyme was stable at between pH 7.5 and 9.5 and at 4 ℃ and 12 ℃. Under the optimal reaction conditions, its Km and Kcat were 0.44 mmol/L and (31.49±2.5) s-1, respectively. With FKP catalyzing at 45 ℃ for 11 h, 2.1 mmol/L of GDP-L-fucose from 10 mmol/L of L-fucose was finally synthesized.
ZHAI Y,HAN D,PAN Y,et al.Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering[J].Enzyme and Microbial Technology,2015,69:38-45.
CHIN Y W,KIM J Y,LEE W H,et al.Enhanced production of 2′-fucosyllactose in engineered Escherichia coli BL21 star(DE3) by modulation of lactose metabolism and fucosyltransferase[J].Journal of Biotechnology,2015,210:107-115.
SANZ S,BANDINI G,OSPINA D,et al.Biosynthesis of GDP-fucose and other sugar nucleotides in the blood stages of plasmodium falciparum[J].Journal of Biological Chemistry,2013,288(23):16 506-16 517.
STURLA L,PUGLIELLI L,TONETTI M,et al.Impairment of the golgi GDP-l-Fucose transport and unresponsiveness to fucose replacement therapy in LAD II patients[J].Pediatric Research,2001,49(4):537-542.
CHIN Y W,KIM J H,SEO N,et al.Metabolic engineering of Escherichia coli to produce 2′-fucosyllactose via salvage pathway of guanosine 5′-diphosphate (GDP)-L-fucose[J].Biotechnology and Bioengineering,2016,113(11):2 443-2 452.
CHENG C,GU J,SU J,et al.Crystallization,preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/L-fucose-1-P-guanylyltransferase from Bacteroides fragilis[J].Acta Crystallographica,2014,70(9):1 206-1 210.
KOTAKE T,HOJO S,TAJIIMA N,et al.A bifunctional enzyme with L-Fucokinase and GDP-L-fucose pyrophosphorylase activities salvages free L-Fucose in Arabidopsis[J].Journal of Biological Chemistry,2008,283(13):8 125-8 135.
COMSTOCK L E,COYNE M J,TZIANABOS A O,et al.Interstrain variation of the polysaccharide B biosynthesis locus of Bacteroides fragilis: characterization of the region from strain 638R[J].Journal of Bacteriology,1999,181(19):6 192-6 196.
PASTUSZAK I,KETCHUM C,HERMANSON G,et al.GDP-L-fucose pyrophosphorylase: purification,cDNA cloning,and properties of the enzyme[J].Journal of Biological Chemistry,1998,273(46):30 165-30 174.
PARK S H,PASTUSZAK I,DRAKE R,et al.Purification to apparent homogeneity and properties of pig kidney L-Fucose kinase[J].Journal of Biological Chemistry,1998,273(10):5 685-5 691.
OHASHI H,WAHL C,OHASHI T,et al.Effective synthesis of guanosine 5’-diphospho-β-L-galactose using Bacterial L-Fucokinase/Guanosine 5’-diphosphate-L-fucose pyrophosphorylase[J].Advanced Synthesis & Catalysis,2017,359(23):4 229-4 234.
WANG W,HU T,FRANTOM P A,et al.Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives[J].Proceedings of the National Academy of Sciences,2009,106(38):16 096-16 101.
SORA Y,LIU Jingjing,JU Y E,et al.Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered Saccharomyces cerevisiae[J].Microbial Cell Factories,2018,17(1):101.
LIU T W,ITO H,CHIBA Y,et al.Functional expression of L-fucokinase/guanosine 5’-diphosphate-L-fucose pyrophosphorylase from Bacteroides fragilis in Saccharomyces cerevisiae for the production of nucleotide sugars from exogenous monosaccharides[J].Glycobiology,2011,21(9):1 228-1 236.
WANG X,YU S,ZHANG T,et al.Identification of a recombinant inulin fructotransferase (difructose dianhydride III forming) from Arthrobacter sp. 161MFSha2.1 with high specific activity and remarkable thermostability[J].Journal of Agricultural and Food Chemistry,2015,63(13):3 509-3 515.
STAUDACHER E,ALTMANN F,WILSON I B H,et al.Fucose in N-glycans: from plant to man[J].Biochimica et Biophysica Acta,1999,1 473(1):230-236.
VILLALOBOS J A,YI B R,WALLACE I S,et al.2-Fluoro-L-Fucose is a metabolically incorporated inhibitor of plant cell wall polysaccharide fucosylation[J].PLOS ONE,2015,10(9):e0139091.
WEICHERT S,JENNEWEIN S,HUFNER,et al.Bioengineered 2′-fucosyllactose and 3-fucosyllactose inhibit the adhesion of Pseudomonas aeruginosa and enteric pathogens to human intestinal and respiratory cell lines[J].Nutrition Research,2013,33(10):831-838.
ISHIHARA H,HEATH E C.The metabolism of L-Fucose[J].Journal of Biological Chemistry,1968,237(8):2 423-2 426.
GALLEGO P,PLANELL R,BENACH J,et al.Structural characterization of the enzymes composing the arginine deiminase pathway in mycoplasma penetrans[J].PLOS ONE, 2012, 7(10):e47886.
KILKER R D,SHUEY D K,SERIF G S.Isolation and properties of porcine thyroid fucokinase[J].BBA-Enzymology,1979,570(2):271-283.