GfsA is a β1,5-galactofuranosyltransferase involved in the biosynthesis of the galactofuran side chain of fungal-type galactomannan in Aspergillus fumigatus

• Published in: Glycobiology (Oxford) 2017-06, Vol.27 (6), p.568-581
• Main Authors: Katafuchi, Yukako, Li, Qiushi, Tanaka, Yutaka, Shinozuka, Saki, Kawamitsu, Yohei, Izumi, Minoru, Ekino, Keisuke, Mizuki, Keiji, Takegawa, Kaoru, Shibata, Nobuyuki, Goto, Masatoshi, Nomura, Yoshiyuki, Ohta, Kazuyoshi, Oka, Takuji
• Format: Article
• Language: English
• Subjects: Aspergillus fumigatus - enzymology; Fungal Polysaccharides - chemistry; Fungal Polysaccharides - metabolism; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Furans - chemistry; Furans - metabolism; Galactosyltransferases - chemistry; Galactosyltransferases - genetics; Galactosyltransferases - metabolism; Manganese - chemistry; Mannans - chemistry; Mannans - metabolism
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwx028

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.