Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides

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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.
Funding Information
  • Ministry of Economy and Competitiveness | Agencia Estatal de Investigación (BFU2017-92223-EXP, SEV-2016-0644)
  • Eusko Jaurlaritza (KK-2019/00076)
  • U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (T32 AI095190, R01 GM096973)
  • U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases

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