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

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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) 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.

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 2.4.1.1 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