Active site flexibility revealed in crystal structures of <i>Parabacteroides merdae</i> β‐glucuronidase from the human gut microbiome

Little, Michael S.; Ervin, Samantha M.; Walton, William G.; Tripathy, Ashutosh; Xu, Yongmei; Liu, Jian; Redinbo, Matthew R.

doi:10.1002/pro.3507

Cited by 18 publications

(11 citation statements)

References 44 publications

(86 reference statements)

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“…Reactions consisted of 5 l of uronate dehydrogenase (1 M final), 5 l of enzyme (30 nM final), 10 l of NAD ϩ (2 mM final), and 30 l of E1-3G or E2-17G (500 M final), all diluted in assay buffer (50 mM HEPES, 50 mM NaCl, various pH) ( Fig. S1) (47,48). The pH of each reaction was chosen based on the optimal pH determined for each GUS with pNPG (28).…”

Section: In Vitro Estrogen-processing Coupled Assaymentioning

confidence: 99%

Gut microbial β-glucuronidases reactivate estrogens as components of the estrobolome that reactivate estrogens

Ervin

Lim

et al. 2019

Journal of Biological Chemistry

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199

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Edited by Wolfgang Peti Gut microbial ␤-glucuronidase (GUS) enzymes have been suggested to be involved in the estrobolome, the collection of microbial reactions involving estrogens. Furthermore, bacterial GUS enzymes within the gastrointestinal tract have been postulated to be a contributing factor in hormone-driven cancers. However, to date, there has been no experimental evidence to support these hypotheses. Here we provide the first in vitro analysis of the ability of 35 human gut microbial GUS enzymes to reactivate two distinct estrogen glucuronides, estrone-3-glucuronide and estradiol-17-glucuronide, to estrone and estradiol, respectively. We show that certain members within the Loop 1, mini-Loop 1, and FMN-binding classes of gut microbial GUS enzymes can reactivate estrogens from their inactive glucuronides. We provide molecular details of key interactions that facilitate these catalytic processes and present the structures of two novel human gut microbial GUS enzymes related to the estrobolome. Further, we demonstrate that estrogen reactivation by Loop 1 bacterial GUS enzymes can be inhibited both in purified enzymes and in fecal preparations of mixed murine fecal microbiota. Finally, however, despite these in vitro and ex vivo data, we show that a Loop 1 GUS-specific inhibitor is not capable of reducing the development of tumors in the PyMT mouse model of breast cancer. These findings validate that gut microbial GUS enzymes participate in the estrobolome but also suggest that the estrobolome is a multidimensional set of processes ongoing within the mammalian gastrointestinal tract that likely involves many enzymes, including several distinct types of GUS proteins.

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Section: In Vitro Estrogen-processing Coupled Assaymentioning

confidence: 99%

Gut microbial β-glucuronidases reactivate estrogens as components of the estrobolome that reactivate estrogens

Ervin

Lim

et al. 2019

Journal of Biological Chemistry

Self Cite

199

157

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“…Inhibition. The hundreds of unique gut microbial GUS enzymes mapped to date have been categorized into six functionally distinct groups based on active site architecture (13)(14)(15)(16)(17)(18)(19)(20). GUSs are encoded by Bacteroidetes, Firmicutes, and Proteobacteria; among these phyla, Proteobacteria are unique in encoding only Loop 1 (L1) GUS orthologs, which are most efficient with smaller glucuronidated substrates (14).…”

Section: Gus Loop Architecture Dictates Sn38-g Processing Efficiency Andmentioning

confidence: 99%

Targeted inhibition of gut bacterial β-glucuronidase activity enhances anticancer drug efficacy

Bhatt

Pellock

Biernat

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial β-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immunedeficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage. microbiome | chemotherapy | cancer | gastrointestinal toxicity I rinotecan (CPT-11) is essential for treating colorectal and pancreatic cancers and is frequently administered as a mixture with 5-fluorouracil and/or oxaliplatin. Ongoing clinical trials seek to extend irinotecan to other forms of cancer. However, irinotecan causes both myelosuppression and gastrointestinal (GI) side effects, including mucositis and late-onset diarrhea, which are often dose limiting: 88% of irinotecan patients develop diarrhea, with 30% experiencing acute grade 3 to 4 diarrhea (1-3). This toxicity is frequently refractory to antimotility drugs. Thus, treating irinotecaninduced diarrhea is a significant clinical need (4, 5).The irinotecan prodrug is activated in vivo to SN38, a potent topoisomerase I inhibitor (6) that retards the growth of rapidly proliferating cells in tumors and the intestinal epithelium, which renews every 5 d. SN38 is detoxified through the addition of glucuronic acid (GlcA) to form SN38-G, a compound marked for elimination via the GI tract. Gut commensal bacterial β-glucuronidase (GUS) proteins remove GlcA from SN38-G. Reactivated SN38 inflicts epithelial damage, resulting in bleeding diarrhea and acute weight loss in animal models (7). We reduced irinotecan-induced gut toxicity using inhibitors that selectively, nonlethally, and potently block the actions of gut bacterial GUS enzymes (8,9). The utility of GUS inhibition also extends to drugs be...

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“…The majority of the point mutations observed were base deletions (40%) and insertions (50%) causing frame shifts, while base substitutions represented the minority (10%). The types of point mutation were independent from time point or strain, and most mutations were found within a range of 500 bp of the gusA gene encoding the catalytic center of the GusA enzyme [ 65 ]. Furthermore, deletions of 62 bp of the gusA gene were found within the 25% N-terminal portion of the GusA protein.…”

Section: Resultsmentioning

confidence: 99%

Towards a 'chassis' for bacterial magnetosome biosynthesis: genome streamlining of Magnetospirillum gryphiswaldense by multiple deletions

et al. 2021

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Background Because of its tractability and straightforward cultivation, the magnetic bacterium Magnetospirillum gryphiswaldense has emerged as a model for the analysis of magnetosome biosynthesis and bioproduction. However, its future use as platform for synthetic biology and biotechnology will require methods for large-scale genome editing and streamlining. Results We established an approach for combinatory genome reduction and generated a library of strains in which up to 16 regions including large gene clusters, mobile genetic elements and phage-related genes were sequentially removed, equivalent to ~ 227.6 kb and nearly 5.5% of the genome. Finally, the fragmented genomic magnetosome island was replaced by a compact cassette comprising all key magnetosome biosynthetic gene clusters. The prospective 'chassis' revealed wild type-like cell growth and magnetosome biosynthesis under optimal conditions, as well as slightly improved resilience and increased genetic stability. Conclusion We provide first proof-of-principle for the feasibility of multiple genome reduction and large-scale engineering of magnetotactic bacteria. The library of deletions will be valuable for turning M. gryphiswaldense into a microbial cell factory for synthetic biology and production of magnetic nanoparticles.

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Active site flexibility revealed in crystal structures of Parabacteroides merdae β‐glucuronidase from the human gut microbiome

Cited by 18 publications

References 44 publications

Gut microbial β-glucuronidases reactivate estrogens as components of the estrobolome that reactivate estrogens

Gut microbial β-glucuronidases reactivate estrogens as components of the estrobolome that reactivate estrogens

Targeted inhibition of gut bacterial β-glucuronidase activity enhances anticancer drug efficacy

Towards a 'chassis' for bacterial magnetosome biosynthesis: genome streamlining of Magnetospirillum gryphiswaldense by multiple deletions

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