Raffinose family oligosaccharide utilisation by probiotic bacteria: insight into substrate recognition, molecular architecture and diversity of GH36 alpha-galactosidases

Maher Abou Hachem, Folmer Fredslund, Joakim Mark Andersen, Rene Jonsgaard Larsen, Avishek Majumder, Morten Ejby Hansen, Gabriella Christina van Zanten, S. J. Lahtinen, R. Barrangou, T. Klaenhammer, S. Jacobsen, P. M. Coutinho, Leila Lo Leggio, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review


    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) alpha-galatosidase encoding genes occur together with sugar transport systems of the glycoside-pentoside-hexuronide cation symporter family (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 homotetrameric alpha-galactosidase from L. acidophilus NCFM (LaMel36A) was determined in complex with galactose bound in the active site to 1.58 angstrom. 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.
    Original languageEnglish
    JournalBiocatalysis and Biotransformation
    Issue number3
    Pages (from-to)316-325
    Publication statusPublished - 2012


    • functional diversity
    • GenBank sequence data
    • molecular architecture
    • structural diversity
    • substrate recognition
    • Actinomycetes and Related Organisms Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Irregular Nonsporing Gram-Positive Rods [08890] Bifidobacterium animalis lactis subspecies strain-Bl-04
    • Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Bacteroidaceae [06901] Thermotoga maritima species
    • Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Regular Nonsporing Gram-Positive Rods [07830] Lactobacillus acidophilus species strain-NCFM
    • Facultatively Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Enterobacteriaceae [06702] Escherichia coli species
    • alpha-galatosidase
    • galactomannan 11078-30-1
    • galactose 26566-61-0
    • genes
    • glycoside hydrolase family 36 GH36
    • Mel36A monomer N-terminal domain, C-terminal domain
    • monosaccharides
    • phosphorylase 9035-74-9 EC
    • probiotic
    • raffinose family oligosaccharides RFO
    • sucrose hydrolases
    • 03502, Genetics - General
    • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
    • 10068, Biochemistry studies - Carbohydrates
    • 10802, Enzymes - General and comparative studies: coenzymes
    • 31000, Physiology and biochemistry of bacteria
    • 31500, Genetics of bacteria and viruses
    • Biochemistry and Molecular Biophysics
    • Enzymology
    • Molecular Genetics


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