title: Design of an α-L-transfucosidase for the synthesis of fucosylated HMOs.
abstract: Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-l-Fucosidases catalyze the hydrolysis of α-l-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α-l-fucosidase from Thermotoga maritima (TmαFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution ( Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-l-transfucosidase starting with the α-l-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon_2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs.
authors.affiliation: Unité de Fonctionnalité et Ingénierie des Protéines (UFIP), Université de Nantes, UMR CNRS 6286, Faculté des Sciences et des Techniques, 2, rue de la Houssinière, BP 92208, NANTES Cedex 3 F- 44322, France.
authors.forename: Amélie
authors.initials: A
authors.lastname: Saumonneau
authors.affiliation: Glycom A/S, Diplomvej 373, Kongens Lyngby 2800, Denmark.
authors.forename: Elise
authors.initials: E
authors.lastname: Champion
authors.affiliation: Glycom A/S, Diplomvej 373, Kongens Lyngby 2800, Denmark.
authors.forename: Pauline
authors.initials: P
authors.lastname: Peltier-Pain
authors.affiliation: Glycom A/S, Diplomvej 373, Kongens Lyngby 2800, Denmark.
authors.forename: Dora
authors.initials: D
authors.lastname: Molnar-Gabor
authors.affiliation: Unité de Fonctionnalité et Ingénierie des Protéines (UFIP), Université de Nantes, UMR CNRS 6286, Faculté des Sciences et des Techniques, 2, rue de la Houssinière, BP 92208, NANTES Cedex 3 F- 44322, France.
authors.forename: Johann
authors.initials: J
authors.lastname: Hendrickx
authors.affiliation: Unité de Fonctionnalité et Ingénierie des Protéines (UFIP), Université de Nantes, UMR CNRS 6286, Faculté des Sciences et des Techniques, 2, rue de la Houssinière, BP 92208, NANTES Cedex 3 F- 44322, France.
authors.forename: Vinh
authors.initials: V
authors.lastname: Tran
authors.affiliation: Glycom A/S, Diplomvej 373, Kongens Lyngby 2800, Denmark.
authors.forename: Markus
authors.initials: M
authors.lastname: Hederos
authors.affiliation: Glycom A/S, Diplomvej 373, Kongens Lyngby 2800, Denmark.
authors.forename: Gyula
authors.initials: G
authors.lastname: Dekany
authors.affiliation: Unité de Fonctionnalité et Ingénierie des Protéines (UFIP), Université de Nantes, UMR CNRS 6286, Faculté des Sciences et des Techniques, 2, rue de la Houssinière, BP 92208, NANTES Cedex 3 F- 44322, France charles.tellier@univ-nantes.fr.
authors.forename: Charles
authors.initials: C
authors.lastname: Tellier
mesh_terms: D000606:Amino Sugars / Q000737:chemistry; D001644:Bifidobacterium / Q000201:enzymology; D005643:Fucose / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007223:Infant; D008895:Milk, Human / Q000737:chemistry*; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000737:chemistry* / Q000235:genetics
chemical_list: D000606:Amino Sugars; D009844:Oligosaccharides; D011134:Polysaccharides; C434666:lacto-N-fucopentaose III; C018991:lacto-N-difucohexaose I; C041762:lacto-N-fucopentaose II; D005643:Fucose; D005644:alpha-L-Fucosidase
keywords: Bifidobacterium longum subsp. infantis; enzymatic synthesis; fucosidase; human milk oligosaccharides; transfucosylation