Microbial glucosinolate metabolism: Characterization of Enterococcus faecalis XL1 and functional analysis of a GH4 enzyme

Glucosinolates (GSLs), sulfur-containing plant metabolites abundant in cruciferous vegetables, are hydrolyzed to isothiocyanates (ITCs) – bioactive compounds with antimicrobial, anticancer, and anti-inflammatory properties. When plant myrosinases are inactivated by cooking, gut microbes become the primary contributors to ITC formation. However, the microbial enzymes and pathways responsible for GSL metabolism remain poorly understood. This dissertation investigates the bacterial mechanisms underlying GSL hydrolysis, focusing primarily on Enterococcus faecalis XL1, a gut isolate obtained from broccoli-fed rats. Whole-genome sequencing and gene expression analyses identified multiple β-glucoside phosphotransferase system (PTS) transporters and glycoside hydrolases (GHs) responsive to sinigrin, with a GH4 enzyme Ef5510, showing the strongest induction. Functional studies revealed that Ef5510 is an NAD+/Mn2+-dependent hydrolase, and the PTS-mediated uptake step represents the rate-limiting stage of the pathway. Heterologous expression and kinetic characterization using a synthesized substrate, p-nitrophenyl-β-D-glucopyranoside-6-phosphate, confirmed Ef5510’s redox-assisted catalytic activity, suggesting a phosphorylation-coupled, GH4-mediated route distinct from classic GH1 systems. Complementary isolation studies from broccoli-fed mice revealed that GSL metabolism is not limited to ITC-forming routes but may also involve alternative pathways, including sulfatase-dependent detoxification. Together, these findings expand the understanding of microbial myrosinase-like activity, highlighting E. faecalis XL1 as a novel model for studying redox-driven GSL metabolism and providing new insights into how gut bacteria contribute to the bioactivation of dietary GSLs.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Food Science & Human Nutrition

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Li, Jiaxuan

Contributors dc:contributor

• Miller, Michael J.
• Banerjee, Pratik
• Jeffery, Elizabeth H.
• Sirk, Shannon

• Glucosinolate metabolism
• Isothiocyanates
• Enterococcus faecalis
• Glycoside hydrolase GH4
• Phosphotransferase system (PTS)
• Gut microbial biotransformation

Statement dc:rights

• Copyright 2025 Jiaxuan Li

Language dc:language

en

Handle dc:identifier

https://hdl.handle.net/2142/132768

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/132768