Journal article
Substrate preference of an ABC importer corresponds to selective growth on β-(1,6)-galactosides in Bifidobacterium animalis subsp. lactis
Department of Biotechnology and Biomedicine, Technical University of Denmark1
Protein Glycoscience and Biotechnology, Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark2
Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark3
University of Copenhagen4
Enzyme and Protein Chemistry, Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark5
Bifidobacteria are exposed to substantial amounts of dietary β-galactosides. Distinctive preferences for growth on different β-galactosides are observed within Bifidobacterium members, but the basis of these preferences remains unclear. We previously described the first β-(1,6)/(1,3)-galactosidase from Bifidobacterium animalis subsp. lactis Bl-04.
This enzyme is relatively promiscuous, exhibiting only 5-fold higher efficiency on the preferred β-(1,6)-galactobiose than the β-(1,4) isomer. Here, we characterize the solute-binding protein (Bal6GBP) that governs the specificity of the ABC transporter encoded by the same β-galactoside-utilization locus.
We observed that although Bal6GBP recognizes both β-(1,6)- and β-(1,4)-galactobiose, Bal6GBP has a 1630-fold higher selectivity for the former, reflected in dramatic differences in growth, with several hours lag on less preferred β-(1,4)- and β-(1,3)-galactobiose. Experiments performed in the presence of varying proportions of β-(1,4)/ β-(1,6)-galactobioses indicated that the preferred substrate was preferentially depleted from the culture supernatant.
This established that the poor growth on the non-preferred β-(1,4) was due to inefficient uptake. We solved the structure of Bal6GBP in complex with β-(1,6)-galactobiose at 1.39 Å resolution, revealing the structural basis of this strict selectivity. Moreover, we observed a close evolutionary relationship with the human milk disaccharide lacto-N-biose-binding protein from Bifidobacterium longum, indicating that the recognition of the non-reducing galactosyl is essentially conserved, whereas the adjacent position is diversified to fit different glycosidic linkages and monosaccharide residues.
These findings indicate that oligosaccharide uptake has a pivotal role in governing selectivity for distinct growth substrates and have uncovered evolutionary trajectories that shape the diversification of sugar-uptake proteins within Bifidobacterium.
Language: | English |
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Publisher: | American Society for Biochemistry and Molecular Biology |
Year: | 2019 |
Pages: | 11701-11711 |
ISSN: | 1083351x , 00219258 and 10678816 |
Types: | Journal article |
DOI: | 10.1074/jbc.RA119.008843 |
ORCIDs: | Theilmann, Mia Christine , Svensson, Birte , Abou Hachem, Maher and 0000-0002-5135-0882 |
ABC transport Actinobacteria Bifidobacteria Crystal structure Enzyme kinetics Galactoogliosaccharides (GOS) Human gut microbiota Human milk ogliosaccharides (HMO) Isothermal titration calorimetry (ITC) Microbiome Prebitoic Probiotic Protein evolution Surface plasmon resonance (SPR)
ABC transporter ATP-Binding Cassette Transporters Amino Acid Sequence Bacterial Proteins Bifidobacterium animalis Binding Sites Catalytic Domain Crystallography, X-Ray Evolution, Molecular Galactosidases Galactosides Kinetics Molecular Dynamics Simulation Protein Binding Substrate Specificity actinobacteria beta-galactoside bifidobacteria crystal structure enzyme kinetics evolution galactooligosaccharides (GOS) human gut microbiota human milk oligosaccharides (HMO) isothermal titration calorimetry (ITC) microbiome prebiotics probiotic protein evolution surface plasmon resonance (SPR)