Mammalian hydroxylation of microbiome-derived obesogen, delta-valerobetaine, to homocarnitine, a 5-carbon carnitine analog

The recently discovered microbiome-generated obesogen, δ-valerobetaine (5-(trimethylammonio)pentanoate), is a 5-carbon structural analog of the carnitine precursor, γ-butyrobetaine. Here, we report that δ-valerobetaine is enzymatically hydroxylated by mammalian γ-butyrobetaine dioxygenase (BBOX) to form 3-hydroxy-5-(trimethylammonio)pentanoate, a 5-carbon analog of carnitine, which we term homocarnitine. Homocarnitine production by human liver extracts depends upon the required BBOX cofactors, 2-oxoglutarate, Fe²⁺, and ascorbate. Molecular dynamics simulations show successful docking of δ-valerobetaine and homocarnitine to BBOX, pharmacological inhibition of BBOX prevents homocarnitine production, and transfection of a liver cell line with BBOX substantially increases production. Furthermore, an in vivo isotope tracer study shows the conversion of ¹³C₃-trimethyllysine to ¹³C₃-δ-valerobetaine then ¹³C₃-homocarnitine in mice, confirming the in vivo production of homocarnitine. Functional assays show that carnitine palmitoyltransferase acylates homocarnitine to acyl-homocarnitine, analogous to the reactions for the carnitine shuttle. Studies of mouse tissues and human plasma show widespread distribution of homocarnitine and fatty acyl-homocarnitines. The respective structural similarities of homocarnitine and acyl-homocarnitines to carnitine and acyl-carnitines indicate that homocarnitine could impact multiple sites of carnitine distribution and activity, potentially mediating microbiome-associated obesity and metabolic disorders.