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

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 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 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

Acknowledgements

Karina Jansen (Technical University of Denmark), Dorthe Boelskifte and Jens-Christian Navarro Poulsen (University of Copenhagen) are thanked for technical assistance.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by a grant from the Danish Strategic Research Council's Programme Committee on Health, Food and Welfare to the project ‘Gene discovery and molecular interactions in prebiotics/probiotics systems. Focus on carbohydrate prebiotics’ as well as the Danish Natural Science Research Council, the Centre for Advanced Food Studies and the Carlsberg Foundation. DANSCATT (Danish National Research Council) and the European Community – Research Infrastructure Action under the FP6 ‘Structuring the European Research Area’ Programme are acknowledged for financial support of synchrotron access. DANISCO A/S is also acknowledged for financial support for PhD student JMA.