title: GfsA is a β1,5-galactofuranosyltransferase involved in the biosynthesis of the galactofuran side chain of fungal-type galactomannan in Aspergillus fumigatus.
abstract: Previously, we reported that GfsA is a novel galactofuranosyltransferase involved in the biosynthesis of O-glycan, the proper maintenance of fungal morphology, the formation of conidia and anti-fungal resistance in Aspergillus nidulans and A. fumigatus (Komachi Y et al., 2013. GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol. Microbiol. 90:1054-1073). In the present paper, to gain an in depth-understanding of the enzymatic functions of GfsA in A. fumigatus (AfGfsA), we established an in vitro assay to measure galactofuranosyltransferase activity using purified AfGfsA, UDP-α-d-galactofuranose as a sugar donor, and p-nitrophenyl-β-d-galactofuranoside as an acceptor substrate. LC/MS, 1H-NMR and methylation analyses of the enzymatic products of AfGfsA revealed that this protein has the ability to transfer galactofuranose to the C-5 position of the β-galactofuranose residue via a β-linkage. AfGfsA requires a divalent cation of manganese for maximal activity and consumes UDP-α-d-galactofuranose as a sugar donor. Its optimal pH range is 6.5-7.5 and its optimal temperature range is 20-30°C. 1H-NMR, 13C-NMR and methylation analyses of fungal-type galactomannan extracted from the ∆AfgfsA strain revealed that AfGfsA is responsible for the biosynthesis of β1,5-galactofuranose in the galactofuran side chain of fungal-type galactomannan. Based on these results, we conclude that AfGfsA acts as a UDP-α-d-galactofuranose: β-d-galactofuranoside β1,5-galactofuranosyltransferase in the biosynthetic pathway of galactomannans.
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Katafuchi
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan.
authors.forename: Qiushi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Infection and Host Defense, Tohoku Medical and Pharmaceutical University, Komatsushima 4-4-1, Sendai 981-8558, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Graduate School of Environmental and Life Science, Okayama University, Tsushimanaka 1-1-1, Okayama 700-8530, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Shinozuka
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Kawamitsu
authors.affiliation: Graduate School of Environmental and Life Science, Okayama University, Tsushimanaka 1-1-1, Okayama 700-8530, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Izumi
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ekino
authors.affiliation: Department of Nanoscience, Faculty of Engineering, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Mizuki
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Takegawa
authors.affiliation: Department of Infection and Host Defense, Tohoku Medical and Pharmaceutical University, Komatsushima 4-4-1, Sendai 981-8558, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Shibata
authors.affiliation: Department of Applied Biochemistry and Food Science, Saga University, Honjo-machi 1, Saga 840-8502, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Goto
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Nomura
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Ohta
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan.
authors.forename: Takuji
authors.initials: T
authors.lastname: Oka
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology*; D062610:Fungal Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005663:Furans / Q000737:chemistry / Q000378:metabolism; D005690:Galactose / Q000031:analogs & derivatives; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008345:Manganese / Q000737:chemistry; D008351:Mannans / Q000737:chemistry / Q000378:metabolism
chemical_list: D062610:Fungal Polysaccharides; D005656:Fungal Proteins; D005663:Furans; D008351:Mannans; C012990:galactomannan; D008345:Manganese; D005700:Galactosyltransferases; C039281:furan; D005690:Galactose
keywords: Aspergillus; cell wall; galactofuranose; galactomannan; glycosyltransferase