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Bacterial β-glucuronidase (GUS) enzymes cause drug toxicity by reversing Phase II glucuronidation in the gastrointestinal tract. While many human gut microbial GUS enzymes have been examined with model glucuronide substrates like p-nitrophenol-β-D-glucuronide (pNPG), the GUS orthologs that are most efficient at processing drug-glucuronides remain unclear. Here we present the crystal structures of GUS enzymes from human gut commensals Lactobacillus rhamnosus, Ruminococcus gnavus, and Faecalibacterium prausnitzii that possess an active site loop (Loop 1; L1) analogous to that found in E. coli GUS, which processes drug substrates. We also resolve the structure of the No Loop GUS from Bacteroides dorei. We then compare the pNPG and diclofenac glucuronide processing abilities of a panel of twelve structurally diverse GUS proteins, and find that the new L1 GUS enzymes presented here process small glucuronide substrates inefficiently compared to previously characterized L1 GUS enzymes like E. coli GUS. We further demonstrate that our GUS inhibitors, which are effective against some L1 enzymes, are not potent towards all. Our findings pinpoint active site structural features necessary for the processing of drug-glucuronide substrates and the inhibition of such processing.

Dissecting membrane dislocation Mistargeting and misinsertion of membrane proteins causes proteostasis stress and dysfunction of intracellular organelles, which can lead to disease. McKenna et al. found that a conserved orphan P-type adenosine triphosphatase (ATPase) transporter removes misinserted transmembrane segments from the endoplasmic reticulum (ER). Functional reconstitutions and cryo–electron microscopy structures show how this ATPase selectively extracts mitochondrial proteins that are mistargeted to the ER and transmembrane segments that are inserted in the wrong orientation. This work identifies polypeptides as a new class of P-type ATPase substrates and defines a new protein quality-control mechanism at the ER. Science , this issue p. eabc5809

Gut microbial beta-glucuronidases are one of the key players in reactivating drug metabolites in the gut lumen. This reactivation can sometimes lead to dose-limiting side effects, such as severe diarrhea and intestinal bleeding. Thus, understanding the structures and functions of these beta-glucuronidases is critical for developing adjuvant therapies to alleviate the gut microbial beta-glucuronidase-induced gastrointestinal toxicities. However, the structural and functional diversities of gut microbial beta-glucuronidase enzyme class are still poorly characterized. In this thesis, we describe three independent studies to understand these diversities. First, we studied the biochemical aspects of Streptococcus Agalactiae beta-glucuronidase and Bacteroides dorei beta-glucuronidase and of structural aspects Bacteroides dorei beta-glucuronidase inhibition. Next, we used loop swap mutants in an attempt to understand the function diversity caused by the extended active site loops. Last, we performed biochemical assays on a previously poorly characterized class of beta-glucuronidase. Our works are a part of a collective effort to explore the gut microbial beta-glucuronidase as an enzyme family. In the future, we can use our knowledge gained from these studies to further our knowledge on the interactions between the gut microbiota and drug metabolites and select beta-glucuronidases as targets for inhibitor design.