THE BIOTRANSFORMATION OF BILE ACIDS BY A COMMON MEMBER OF THE BISON GUT MICROBIOME
Date
2025-05-15
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Thesis
Degree Level
Doctoral
Abstract
Bacterial biotransformation of bile acids (BAs) is increasingly recognized to play an important role in the health and disease of the host. Bile acid-modifying bacteria participate in the production of a large number of secondary BAs (2 BAs) with known and yet to be discovered activities. In the course of isolating and characterizing bison-associated bacteria, Arthrobacter spp. were routinely found to modify bile acids in vitro. Whole genome sequencing of a single isolate, A. citreus EINP1, revealed the presence of a bile salt hydrolase (BSH) and additional homologues with established roles in bile acid transformation reactions. Characterization of the A. citreus EINP1 BSH (BSHAc) was complete, including the first robust kinetic characterization of any member of this enzyme family with a pan-family inhibitor. BSHAc showed optimum activity at ~57 °C and between pH 5 and 6. Steady-state kinetic characterization of BSHAc using glycine and taurine conjugates of cholic (CA) and deoxycholic acid (DCA) revealed it was a so-called broad spectrum BSH with preference for glycine-conjugates and DCA. Single crystal X-ray diffraction experiments resulted in 2 and 1.6 Å resolution structures of apo- and covalent AAA-10 inhibitor bound forms. The AAA-10 molecules, which contain an -fluoromethylketone (FMK) warhead, was demonstrated to be a slow covalent inhibitor of the enzyme with an inactivation efficiency (kinact/KI) of 24 ± 4 M-1s-1. Further studies confirmed that Arthrobacter spp. capable of modifying BAs are common members of the bison gut microbiome, and the study of A. citreus revealed that the transformation of CA and DCA proceeds through the understudied and incomplete ∆4,6-variant of the 9,10-seco pathway of bile acid metabolism. Twelve distinct biotransformation products of CA and DCA were isolated, and their structures were identified by nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS), and X-ray diffraction experiments. Importantly, metabolites produced by A. citreus EINP1 in vitro were also consistently observed in bison fecal samples, suggesting they play a yet to be determined biological role in the animal.�
Description
Keywords
hydrolase, bile acid, bacteria, enzyme structure, enzyme kinetics, enzyme inhibitor, gut microbiome, biotransformation
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Veterinary Microbiology
Program
Veterinary Microbiology