Gut microbial β-glucuronidases (GUSs) inhibition is a new approach for managing some diseases and medication therapy. However, the structural and functional complexity of GUSs have posed tremendous challenges to discover GUSs inhibitors using Escherichia coli GUS (EcoGUS) alone. This study assessed the effects of twenty-one flavones employing three Loop 1-type GUSs, the main GUSs involved in deglucuronidation of small molecules, on p-nitrophenyl-β-D-glucuronide hydrolysis and a structure-activity relationship is proposed based on in vitro assays and docking study. EcoGUS and Staphylococcus pasteuri GUS showed largely similar inhibition propensities with potencies positively correlating with the total hydroxyl groups and those at ring B of flavones, while docking studies revealed strong interactions developed via ring A and/or C. Streptococcus agalactiae GUS (SagaGUS) displayed
distinct late-onset inhibition and steep dose-response profiles with most tested
compounds. The α-helix in its loop 1 region caused spatial hindrance but offered a hydrophobic surface for contacting with the carbonyl group of flavones is believed to be essential for the allosteric inhibition of SagaGUS. Taken together, the study revealed varied preferences for GUSs belonging to Loop-1 type, highlighting the necessity of adopting multi-GUSs for screening broad-spectrum GUSs inhibitors or tailoring the inhibition based on specific GUS structure.