Structural and functional analysis of a bile salt hydrolase from the bison microbiome

Sep 14, 2024The Journal of biological chemistry

Structure and function of a bile salt breaking enzyme from bison gut bacteria

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Abstract

Second-order rate constants for BSH-catalyzed reactions varied by ∼30-fold, ranging from 1.4 × 10 to 4.3 × 10 Ms.

Bile salt hydrolases (BSHs) are important enzymes in animal microbiomes, involved in processing bile salts.
BSH from Arthrobacter citreus shows broad-spectrum activity, favoring glycine-conjugates and deoxycholic acid (DCA).
A pan-BSH inhibitor, AAA-10, was identified as a slow irreversible inhibitor of BSH, with a rate of inactivation around 2 h.
Structural analysis revealed AAA-10 covalently modifies the enzyme's N-terminal cysteine, providing insight into the inhibition mechanism.
Comparative structural analysis indicates variability in the steroid-binding site among different BSHs, suggesting evolutionary adaptation.

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The bile salt hydrolases (BSHs) are significant constituents of animal microbiomes. An evolving appreciation of their roles in health and disease has established them as targets of pharmacological inhibition. These bacterial enzymes belong to the N-terminal nucleophile superfamily and are best known to catalyze the deconjugation of glycine or taurine from bile salts to release bile acid substrates for transformation and or metabolism in the gastrointestinal tract. Here, we identify and describe the BSH from a common member of the Plains bison microbiome, Arthrobacter citreus (BSH). Steady-state kinetic analyses demonstrated that BSHis a broad-spectrum hydrolase with a preference for glycine-conjugates and deoxycholic acid (DCA). Second-order rate constants (k/K) for BSH-catalyzed reactions of relevant bile salts-glyco- and tauro-conjugates of cholic acid and DCA- varied by ∼30-fold and measured between 1.4 × 10and 4.3 × 10 Ms. Interestingly, a pan-BSH inhibitor named AAA-10 acted as a slow irreversible inhibitor of BSHwith a rate of inactivation (k) of ∼2 hand a second order rate constant (k/K) of ∼24 Msfor the process. Structural characterization of BSHreacted with AAA-10 showed covalent modification of the N-terminal cysteine nucleophile, providing molecular details for an enzyme-stabilized product formed from this mechanism-based inhibitor's α-fluoromethyl ketone warhead. Structural comparison of the BSHs and BSH:inhibitor complexes highlighted the plasticity of the steroid-binding site, including a flexible loop that is variable across well-studied BSHs. Ac Ac cat M Ac Ac inact inact I Ac5 6 -1 -1 -1 -1 -1

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Structural and functional analysis of a bile salt hydrolase from the bison microbiome

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