LC-MS Data for the Nature Communications publication, "SpiR is a gut microbial enzyme that drives cholesterol conversion"

The gut microbiota contributes to cholesterol homeostasis by converting cholesterol into coprostanol, a non-absorbable sterol excreted in the feces. However, the enzymes mediating this process remain poorly defined. Here, we identify spiR, a steroid Δ5-4 isomerase/3-keto reductase from Eubacterium coprostanoligenes that catalyzes the initial oxidation of cholesterol to cholestenone, a requisite step in coprostanol production. We confirm that SpiR oxidizes both cholesterol and pregnenolone, and stereospecifically reduces 3-keto-steroids to 3β-hydroxylated forms. We show that SpiR preferentially binds to cholesterol over related steroids and functions as an NAD(H)-dependent homodimer. Through phylogenetic analysis, we show that spiR clusters with known Δ5-4 isomerases and is restricted to an uncultured clade within Acutalibacteraceae, where it frequently co-occurs with species encoding ismA, a gene previously implicated in cholesterol conversion. We analyze a multi-omic dataset from three human cohorts and find that spiR homologs were strongly enriched in individuals exhibiting cholesterol conversion. We also show that spiR homologs have a greater predictive power for cholesterol conversion than ismA homologs, establishing them as superior markers of microbial cholesterol metabolism. Our findings refine the enzymatic model of cholesterol metabolism in the gut and establish spiR as a critical biomarker and mechanistic driver for microbiome-mediated cholesterol reduction.