# Human gut microbes express functionally distinct endoglycosidases to metabolize the same N-glycan substrate

## TL;DR

Human gut microbes use different enzymes to break down the same sugar structure in the gut, each optimized for specific conditions.

## Contribution

Discovery of a distinct endoglycosidase in Bacteroides thetaiotaomicron with unique functional properties for N-glycan metabolism.

## Key findings

- BT1285 has higher affinity and faster hydrolysis of HM N-glycans compared to BT3987.
- BT1285 and BT3987 function optimally under different environmental conditions.
- Alistipes finegoldii also uses condition-specific endoglycosidases for HM N-glycan processing.

## Abstract

Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-β-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.

The human gut microbiome has a substantial impact on human health. Here, the authors find that prominent human gut microbes express functionally distinct surface endo-β-N-acetylglucosaminidases encoded by different polysaccharide utilization loci to optimally metabolize the same oligomannose N-glycan substrate in the gastrointestinal tract.