Structural basis for the regulation of β-glucuronidase expression by human gut Enterobacteriaceae.

Abstract

The gut microbiota harbor diverse β-glucuronidase (GUS) enzymes that liberate glucuronic acid (GlcA) sugars from small-molecule conjugates and complex carbohydrates. However, only the Enterobacteriaceae family of human gut-associated Proteobacteria maintain a GUS operon under the transcriptional control of a glucuronide repressor, GusR. Despite its potential importance in <i>Escherichia</i>, <i>Salmonella</i>, <i>Klebsiella</i>, <i>Shigella</i>, and <i>Yersinia</i> opportunistic pathogens, the structure of GusR has not been examined. Here, we explore the molecular basis for GusR-mediated regulation of GUS expression in response to small-molecule glucuronides. Presented are 2.1-Å-resolution crystal structures of GusRs from <i>Escherichia coli</i> and <i>Salmonella enterica</i> in complexes with a glucuronide ligand. The GusR-specific DNA operator site in the regulatory region of the <i>E. coli</i> GUS operon is identified, and structure-guided GusR mutants pinpoint the residues essential for DNA binding and glucuronide recognition. Interestingly, the endobiotic estradiol-17-glucuronide and the xenobiotic indomethacin-acyl-glucuronide are found to exhibit markedly differential binding to these GusR orthologs. Using structure-guided mutations, we are able to transfer <i>E. coli</i> GusR's preferential DNA and glucuronide binding affinity to <i>S. enterica</i> GusR. Structures of putative GusR orthologs from GUS-encoding Firmicutes species also reveal functionally unique features of the Enterobacteriaceae GusRs. Finally, dominant-negative GusR variants are validated in cell-based studies. These data provide a molecular framework toward understanding the control of glucuronide utilization by opportunistic pathogens in the human gut.