ORIGINAL ARTICLE Raffinose family oligosaccharide utilisation by probiotic bacteria: insight into substrate recognition, molecular architecture and diversity of GH36 α-galactosidases MAHER ABOU HACHEM 1 , F. FREDSLUND 1 , J. M. ANDERSEN 1 , R. JONSGAARD LARSEN 1 , A. MAJUMDER 1 , M. EJBY 1 , G. VAN ZANTEN 1 , S. J. LAHTINEN 2 , R. BARRANGOU 3 , T. KLAENHAMMER 4 , S. JACOBSEN 1 , P. M. COUTINHO 5 , L. LO LEGGIO 6 & BIRTE SVENSSON 1 1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark, 2 DuPont Nutrition and Health, Sokeritehtaantie 20, FI-02460 Kantvik, Finland, 3 DuPont Nutrition and Health, 3329 Agriculture Drive, Madison,WI 53716, USA, 4 Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Box 7624, Raleigh, NC 27695, USA, 5 Architecture et Fonction des Macromolecules Biologiques, CNRS, Université d’Aix Marseille, Case 932, 163 Avenue de Luminy, 13288, Marseille cedex 9, France, and 6 Biophysical Chemistry Group, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark Abstract The organisation of genes conferring utilisation of raffinose family oligosaccharides (RFOs) has been analysed in several probiotic bacteria from the Bifidobacterium and Lactobacillus genera. Glycoside hydrolase family 36 (GH36) α-galatosidase encoding genes occur together with sugar transport systems of the glycoside–pentoside–hexuronide cation symporter fam- ily (GPH), sugar phosphotransferase systems (PTSs) or ATP-binding cassette systems (ABCs) highlighting the diversity of RFO uptake. The GH36 genes are often clustered together with sucrose hydrolases or phosphorylases ensuring the degradation of RFO to monosaccharides. Differential proteomics and transcriptomics data from our laboratories implicated ABC transporters in the uptake of RFO in both Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Interestingly, only one of three GH36 encoding genes in B. animalis subsp. lactis Bl-04 was upregulated upon growth on RFO, suggesting that the other two gene products may have different specificities. The structure of the GH36 homote- trameric α-galactosidase from L. acidophilus NCFM ( LaMel36A) was determined in complex with galactose bound in the active site to 1.58 Å. Differences in the N- and C-terminal domains of the LaMel36A monomer distinguished it from the monomeric TmGalA from Thermotoga maritima providing a structural rationale for the observed difference in oligomeric states of the two enzymes. Tetramerisation of LaMel36A creates a narrow and deep active site pocket between three monomers, which explains the preference of tetrameric GH36 enzymes for RFO and their lack of activity on polymeric galacto(gluco)mannan. Finally, GH36 was divided into four subgroups based on active site motifs, which illuminates functional and structural diversity in the family and aids further annotation of emerging sequences. Keywords: prebiotic, probiotic, tetrameric α-galactosidases, raffinose, clan GH-D Correspondence: Maher Abou Hachem, Enzyme and Protein Chemistry, DTU Systems Biology, Technical University of Denmark, Søltofts Plads, Bldg. 224, DK-2800 Kgs. Lyngby, Denmark. E-mail: maha@bio.dtu.dk Introduction α-Galactosidases ( α- D-galactoside galactohydrolase; E.C. 3.2.1.22; α-Gal) are exo-acting enzymes that catalyse the removal of terminal α- D-linked galactosyl residues from the non-reducing ends of a variety of substrates and play important roles in health and industrial applications. In lysosomes, α-galactosidase activity is necessary for the turnover of α-galactosides, for example, oligosaccharides, glycoproteins and gly- colipids, and the accumulation of these molecules causes Fabry disease (Garman & Garboczi 2004). In plants, α-Gals are involved in the metabolism of galactoglucomannans and galactomannans, which are structural and storage polysaccharides, respectively, Biocatalysis and Biotransformation, 2012; Early Online: 1–10 ISSN 1024-2422 print/ISSN 1029-2446 online © 2012 Informa UK, Ltd. DOI: 10.3109/10242422.2012.674717