Abstract
The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-β-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine. Here, we provide structural snapshots of EndoBT-3987, including the unliganded form, the EndoBT-3987-Man9GlcNAc2Asn substrate complex, and two EndoBT-3987-Man9GlcNAc and EndoBT-3987-Man5GlcNAc product complexes. In combination with alanine scanning mutagenesis and activity measurements we unveil the molecular mechanism of HM-type recognition and specificity for EndoBT-3987 and an important group of the GH18 ENGases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.
Highlights
The human gut microbiota plays a central role in regulating the metabolism of nutrients and promoting immune homeostasis, immune responses and protection against pathogen colonization
This structure represents an example of an enzyme-substrate complex in the glycoside hydrolase family 18 (GH18) ENGase family
To further advance the understanding of EndoBT-3987 N-glycan specificity, we performed a structural analysis in the context of the GH18 family of ENGases
Summary
The human gut microbiota plays a central role in regulating the metabolism of nutrients and promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. The genomes of Bacteroidetes contain polysaccharide utilization loci (PULs)[12] that encode the apparatus required to utilize multi-subunit carbohydrates, with each PUL orchestrating the degradation of a specific glycan. The Sus-like systems are widespread among the Bacteroidetes members accounting, in many species, for up to one-fifth of their genomes to encode Suslike pathways, as in Bacteroides thetaiotaomicron[14,15,16]. B. thetaiotaomicron encodes a high-mannose mammalian N-glycan (HMNG) depolymerizing system, comprised by four enzymes and two surface glycan binding proteins[7]. According to the currently accepted model, the released HM is held on the surface of B. thetaiotaomicron through the mannose-binding protein BT3986, while
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