Dataset Information

Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum.

ABSTRACT: Infant gut-associated bifidobacteria has a metabolic pathway that specifically utilizes lacto-N-biose I (Gal-?1,3-GlcNAc) and galacto-N-biose (Gal-?1,3-GalNAc) from human milk and mucin glycans. UDP-glucose 4-epimerase (GalE) from Bifidobacterium longum (bGalE) catalyzes epimerization reactions of UDP-Gal into UDP-Glc and UDP-GalNAc into UDP-GlcNAc with the same level of activity that is required to send galacto-hexoses into glycolysis. Here, we determined the crystal structures of bGalE in three ternary complex forms: NAD+/UDP, NAD+/UDP-GlcNAc, and NAD+/UDP-Glc. The broad specificity of bGalE was explained by structural features of the binding pocket for the N-acetyl or C2 hydroxy group of the substrate. Asn200 is located in a pocket of the C2 group, and its side chain adopts different conformations in the complex structures with UDP-Glc and UDP-GlcNAc. On the other side, Cys299 forms a large pocket for the C5 sugar ring atom. The flexible C2 pocket and the large C5 pocket of bGalE are suitable for accommodating both the hydroxy and N-acetyl groups of the substrate during sugar ring rotation in the catalytic cycle. The substrate specificity and active site structure of bGalE were distinct from those of Esherichia coli GalE but similar to those of human GalE.

SUBMITTER: Nam YW

PROVIDER: S-EPMC6668579 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum.

Nam Young-Woo YW Nishimoto Mamoru M Arakawa Takatoshi T Kitaoka Motomitsu M Fushinobu Shinya S

Scientific reports 20190731 1

Infant gut-associated bifidobacteria has a metabolic pathway that specifically utilizes lacto-N-biose I (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and mucin glycans. UDP-glucose 4-epimerase (GalE) from Bifidobacterium longum (bGalE) catalyzes epimerization reactions of UDP-Gal into UDP-Glc and UDP-GalNAc into UDP-GlcNAc with the same level of activity that is required to send galacto-hexoses into glycolysis. Here, we determined the crystal structures of bGalE in thre ...[more]

PMID: 31366978

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Project description:Bifidobacterium longum subsp. infantis is a representative and dominant species in the infant gut and is considered a beneficial microbe. This organism displays multiple adaptations to thrive in the infant gut, regarded as a model for human milk oligosaccharides (HMOs) utilization. These carbohydrates are abundant in breast milk and include different molecules based on lactose. They contain fucose, sialic acid, and N-acetylglucosamine. Bifidobacterium metabolism is complex, and a systems view of relevant metabolic pathways and exchange metabolites during HMO consumption is missing. To address this limitation, a refined genome-scale network reconstruction of this bacterium is presented using a previous reconstruction of B. infantis ATCC 15967 as a template. The latter was expanded based on an extensive revision of genome annotations, current literature, and transcriptomic data integration. The metabolic reconstruction (iLR578) accounted for 578 genes, 1,047 reactions, and 924 metabolites. Starting from this reconstruction, we built context-specific genome-scale metabolic models using RNA-seq data from cultures growing in lactose and three HMOs. The models revealed notable differences in HMO metabolism depending on the functional characteristics of the substrates. Particularly, fucosyl-lactose showed a divergent metabolism due to a fucose moiety. High yields of lactate and acetate were predicted under growth rate maximization in all conditions, whereas formate, ethanol, and 1,2-propanediol were substantially lower. Similar results were also obtained under near-optimal growth on each substrate when varying the empirically observed acetate-to-lactate production ratio. Model predictions displayed reasonable agreement between central carbon metabolism fluxes and expression data across all conditions. Flux coupling analysis revealed additional connections between succinate exchange and arginine and sulfate metabolism and a strong coupling between central carbon reactions and adenine metabolism. More importantly, specific networks of coupled reactions under each carbon source were derived and analyzed. Overall, the presented network reconstruction constitutes a valuable platform for probing the metabolism of this prominent infant gut bifidobacteria.IMPORTANCEThis work presents a detailed reconstruction of the metabolism of Bifidobacterium longum subsp. infantis, a prominent member of the infant gut microbiome, providing a systems view of its metabolism of human milk oligosaccharides.

Project description:In order to gain insight into the effects of human breast milk on the development of the intestinal bifidobacteria and associated health effects, the transcriptome of Bifidobacterium longum LMG 13197 grown in breast milk and formula milk containing galactooligosaccharides (GOS) and long-chain fructooligosaccharides was compared to that obtained in a semisynthetic medium with glucose. Total RNA was isolated from exponentially growing cells and hybridized to a clone library-based microarray. Inserts of clones with significant hybridization signals were sequenced and identified. The B. longum transcriptomes obtained during growth on human and formula milk were more similar to each other than to that obtained from growth in semisynthetic medium with glucose. Remarkably, there were only a few genes implicated in carbohydrate metabolism that were similarly upregulated during growth in both human and formula milk although oligosaccharides were added to the formula. Common highly upregulated genes notably included putative genes for cell surface type 2 glycoprotein-binding fimbriae that are implicated in attachment and colonization in the intestine. Genes involved in carbohydrate metabolism formed the dominant group specifically upregulated in breast milk and included putative genes for N-acetylglucosamine degradation and for metabolism of mucin and human milk oligosaccharides via the galactose/lacto-N-biose gene cluster. This supports the notion that the bifidogenic effect of human milk is to a great extent based on its oligosaccharides. The transcriptional effect of semisynthetic medium containing GOS, which, like human milk, contains a large amount of lactose and galactose, on the B. longum transcriptome was also studied and revealed substantial similarity with carbohydrate-utilization genes upregulated during growth in human milk. This knowledge provides leads to optimizing formula milk to better simulate the observed bifidogenic effects of human breast milk.

Dataset Information

Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum.

Publications

Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum.

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