Aspergillus nidulansα-galactosidase of glycoside hydrolase family 36 catalyses the formation of α-galacto-oligosaccharides by transglycosylation

• Published in: The FEBS journal Vol. 277; no. 17; pp. 3538 - 3551
• Main Authors: Nakai, Hiroyuki, Baumann, Martin J, Petersen, Bent O, Westphal, Yvonne, Hachem, Maher Abou, Dilokpimol, Adiphol, Duus, Jens Ø, Schols, Henk A, Svensson, Birte
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.09.2010; Blackwell Publishing Ltd
• Subjects: acceptor specificity; alpha-Galactosidase - biosynthesis; alpha-Galactosidase - isolation & purification; alpha-Galactosidase - metabolism; Amino Acid Sequence; Aspergillus nidulans - enzymology; Biocatalysis; Carbohydrate Conformation; carbohydrate structural analysis; Cloning, Molecular; Escherichia coli - metabolism; Glycosylation; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Models, Molecular; Oligosaccharides - biosynthesis; Oligosaccharides - chemistry; Phylogeny; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Temperature; transglycosylation; α-galacto-oligosaccharides; α-galactosidase
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2010.07763.x

The α-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic α-galactosidases and α-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L⁻¹ culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with α-(1[rightward arrow]6) regioselectivity from 40 m m 4-nitrophenol α- d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenol α- d-Galp-(1[rightward arrow]6)- d-Galp, α- d-Galp-(1[rightward arrow]6)-α- d-Galp-(1[rightward arrow]6)- d-Glcp and α- d-Galp-(1[rightward arrow]6)-α- d-Galp-(1[rightward arrow]6)- d-Glcp-(α1[rightward arrow]β2)- d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 m m) and the donor 4-nitrophenol α- d-galactopyranoside (40 m m), α-(1[rightward arrow]6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39-58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l-arabinose, l-fucose and l-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l-rhamnose. Structural modelling using Thermotoga maritima GH36 α-galactosidase as the template and superimposition of melibiose from the complex with human GH27 α-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 m m), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred α-galactosyl to 6-OH of the terminal residue in the α-linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the β-linked lactose, lactulose and cellobiose in 28-38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. α- d-Galp-(1[rightward arrow]6)- d-Manp; α- d-Galp-(1[rightward arrow]6)-β- d-Glcp-(1[rightward arrow]4)- d-Glcp; α- d-Galp-(1[rightward arrow]6)-β- d-Galp-(1[rightward arrow]4)- d-Fruf; α- d-Galp-(1[rightward arrow]6)- d-Glcp-(α1[rightward arrow]α1)- d-Glcp; and α- d-Galp-(1[rightward arrow]6)-α- d-Glcp-(1[rightward arrow]3)- d-Fruf.