Discovery and mechanistic characterization of a probiotic-origin 3β-OH-Δ ^5-6 -cholesterol-5β-reductase directly converting cholesterol to coprostanol

On the one hand, cholesterol is a foundational molecule for various structural and biochemical pathways, while elevated cholesterol levels are associated with cardiovascular diseases. Some selected strains of Lactobacilli are also known for modulating cholesterol levels. However, the molecular mechanism of cholesterol transformation by lactobacilli has remained challenging to reveal. This study reports the discovery and role of a microbial 3β-OH-Δ ^5-6 -cholesterol-5β-reductase from Limosilactobacillus fermentum NKN51, which directly converts cholesterol to coprostanol, thereby resolving the enigma. Protein engineering of the reductase enzyme identified the cholesterol and NADP ⁺ interacting amino acids, detailing the catalytic mechanism of 5βChR. Phylogenetic studies emphasize the abundance of 5βChRs in gut commensal lactobacilli, which shares a common ancestor with plant 5β reductases. Meta-analysis results of healthy participant microbiomes underline the significance of 5βChR homologs, while a cohort study reveals an association between higher 5βChR abundance and diabetes. The discovery of the 5βChR enzyme and its molecular mechanism in cholesterol metabolism pave the way for a better understanding of the gut-associated microbiome and the design of practical applications to ameliorate dyslipidemia.