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

Samantha M Ervin,Hao Li,Lauren Lim,Lee R Roberts,Xue Liang,Sridhar Mani,Matthew R Redinbo

doi:10.1074/jbc.ra119.010950

Abstract

Gut microbial β-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 on-going within the mammalian gastrointestinal tract that likely involves many enzymes, including several distinct types of GUS proteins.

Highlights

Gut microbial ␤-glucuronidase (GUS) enzymes have been suggested to be involved in the estrobolome, the collection of microbial reactions involving estrogens
To gain greater insight into the specific sequence-structure-function relationships among GUS proteins, we generated a sequence similarity network (SSN) using sequences of ␤-glucuronidase enzymes found within the Human Microbiome Project (HMP)
Using a multiple-sequence alignment of all 279 GUS enzymes identified from the HMP, we found that 14 contained a loop insert of roughly 25 residues, like in Eubacterium eligens GUS (6BJW) [29], that were near the active site, adjacent to the Loop 1/miniLoop 1 site, that could potentially contribute to binding (Fig. S3)

Summary

Introduction

Gut microbial ␤-glucuronidase (GUS) enzymes have been suggested to be involved in the estrobolome, the collection of microbial reactions involving estrogens. 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 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 and suggest that the estrobolome is a multidimensional set of processes on-going within the mammalian gastrointestinal tract that likely involves many enzymes, including several distinct types of GUS proteins. Upon entry into the GI tract, they are exposed to GUS enzymes that could, in theory, cleave the sugar moiety, reactivating the parent compound and allowing the unconjugated estrogen to be reab-

Results

Discussion

Conclusion