Structure-function studies on drug-reactivating β-glucuronidase from mucin-degrading gut symbiont <i>Akkermansia muciniphila</i>

Gut microbial β-glucuronidases (mGUS) not only regulate several hormones and neurotransmitters, they also impact the efficacy and toxicity of xenobiotics. On certain anticancer drugs, e.g. irinotecan (SN-38), mGUS activity leads to enterohepatic recirculation resulting into severe diarrhea. Here, we report the expression, purification and characterization of AmGUS, a novel β-glucuronidase from <i>Akkermansia muciniphila. A. muciniphila</i> is a prominent gut symbiont with beneficial effects on metabolic health and gut homeostasis. AmGUS demonstrates specificity towards glucuronide substrates with no glucosidase or galactosidase activity. Interestingly, it also shows efficient cleavage of the glucuronidated form of the anti-cancer drug, SN38, potentially leading to its enterohepatic recirculation. Furthermore, we find that AmGUS functions as a monomer, contrary to other GUS enzymes that exist as oligomers. mGUS are classified into distinct loop-types based on different active site loops around a conserved core providing substrate specificity. Computational modeling of AmGUS structure reveals that it belongs to the mL2 loop-type GUS enzymes, despite sharing significant sequence/structural similarity with the mL1 loop-type and NL-type GUSs. Interestingly, AmGUS also has a unique N-terminal loop previously not observed in any other GUS enzyme possibly aiding in the processing of drug-glucuronides. Together, these findings suggest that GUS from <i>A. muciniphila</i> belongs to a new class of GUS enzymes with unique active site loop structures. The presence of this unique GUS enzyme may help <i>A. muciniphila</i> in colonizing the human gut. Overall, this study broadens our knowledge of the structural and functional understanding of GUSome in the human gut microbiome.