Structural basis for substrate-induced activation of 3-hydroxybutyryl-CoA dehydrogenase from Faecalibacterium prausnitzii L2-6

Dysbiosis of the gut microbiota has increasingly been associated with atopic dermatitis (AD), a chronic inflammatory dermatological disorder. Butyrate, a short-chain fatty acid recognized for its significant anti-inflammatory capabilities, has garnered particular interest among gut microbial metabolites. The process that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which is important for making butyrate, is carried out by L26HBD, an enzyme from F. prausnitzii L2-6 that depends on NAD⁺. We determined the crystal structure of L26HBD in association with NAD⁺ and acetoacetyl-CoA to clarify the structural information of its catalytic action. The monomeric enzyme consists of two distinct domains: a C-terminal domain responsible for dimerization and an N-terminal Rossmann fold that binds NAD⁺. The enclosure of the active site arises from a significant conformational shift in the clamp-lid domain induced by substrate binding, with a root-mean-square deviation of 2.88 Å. The induced fit mechanism was corroborated by structural comparisons between the ligand-free and substrate-bound forms, revealing substrate-driven cavity contraction. Despite the identification of the acetoacetyl-CoA binding mechanism, electron density and B-factor measurements indicated that it exhibited lower stability compared to NAD⁺ binding. These findings enhance our understanding of butyrate biosynthesis in commensal gut bacteria by providing mechanistic insights into substrate detection and catalysis by L26HBD.