Chemoproteomic profiling of substrate specificity in gut microbiota-associated bile salt hydrolases.

The gut microbiome possesses numerous biochemical enzymes that biosynthesize metabolites that impact human health. Bile acids comprise a diverse collection of metabolites that have important roles in metabolism and immunity. The gut microbiota-associated enzyme that is responsible for the gateway reaction in bile acid metabolism is bile salt hydrolase (BSH), which controls the host's overall bile acid pool. Despite the critical role of these enzymes, the ability to profile their activities and substrate preferences remains challenging due to the complexity of the gut microbiota, whose metaproteome includes an immense diversity of protein classes. Using a systems biochemistry approach employing activity-based probes, we have identified gut microbiota-associated BSHs that exhibit distinct substrate preferences, revealing that different microbes contribute to the diversity of the host bile acid pool. We envision that this chemoproteomic approach will reveal how secondary bile acid metabolism controlled by BSHs contributes to the etiology of various inflammatory diseases.

Electrostatic Interactions Dictate Bile Salt Hydrolase Substrate Preference.

The human intestines are colonized by trillions of microbes, comprising the gut microbiota, which produce diverse small molecule metabolites and modify host metabolites, such as bile acids, that regulate host physiology. Biosynthesized in the liver, bile acids are conjugated with glycine or taurine and secreted into the intestines, where gut microbial bile salt hydrolases (BSHs) deconjugate the amino acid to produce unconjugated bile acids that serve as precursors for secondary bile acid metabolites. Among these include a recently discovered class of microbially conjugated bile acids (MCBAs), wherein alternative amino acids are conjugated onto bile acids. To elucidate the metabolic potential of MCBAs, we performed detailed kinetic studies to investigate the preference of BSHs for host-conjugated bile acids and MCBAs. We identified a BSH that exhibits positive cooperativity uniquely for MCBAs containing an aromatic side chain. Further molecular modeling and phylogenetic analyses indicated that the BSH preference for aromatic MCBAs is due to a substrate-specific cation-π interaction and is predicted to be widespread among human gut microbial BSHs.

Chemoproteomic Approaches for Unraveling Prokaryotic Biology.

Bacteria are ubiquitous lifeforms with important roles in the environment, biotechnology, and human health. Many of the functions that bacteria perform are mediated by proteins and enzymes, which catalyze metabolic transformations of small molecules and modifications of proteins. To better understand these biological processes, chemical proteomic approaches, including activity-based protein profiling, have been developed to interrogate protein function and enzymatic activity in physiologically relevant contexts. Here, chemoproteomic strategies and technological advances for studying bacterial physiology, pathogenesis, and metabolism are discussed. The development of chemoproteomic approaches for characterizing protein function and enzymatic activity within bacteria remains an active area of research, and continued innovations are expected to provide breakthroughs in understanding bacterial biology.

Electrostatic Interactions Dictate Bile Salt Hydrolase Substrate Preference.

The human intestines are colonized by trillions of microbes, comprising the gut microbiota, which produce diverse small molecule metabolites and modify host metabolites, such as bile acids, that regulate host physiology. Biosynthesized in the liver, bile acids are conjugated with glycine or taurine and secreted into the intestines, where gut microbial bile salt hydrolases (BSHs) deconjugate the amino acid to produce unconjugated bile acids that serve as precursors for secondary bile acid metabolites. Among these include a recently discovered class of microbially-conjugated bile acids (MCBAs), wherein alternative amino acids are conjugated onto bile acids. To elucidate the metabolic potential of MCBAs, we performed detailed kinetic studies to investigate the preference of BSHs for host- and microbially-conjugated bile acids. We identified a BSH that exhibits positive cooperativity uniquely for MCBAs containing an aromatic sidechain. Further molecular modeling and phylogenetic analyses indicated that BSH preference for aromatic MCBAs is due to a substrate-specific cation-π interaction and is predicted to be widespread among human gut microbial BSHs.

Native mass spectrometry-based metabolomics identifies metal-binding compounds.

Although metals are essential for the molecular machineries of life, systematic methods for discovering metal-small molecule complexes from biological samples are limited. Here, we describe a two-step native electrospray ionization-mass spectrometry method, in which post-column pH adjustment and metal infusion are combined with ion identity molecular networking, a rule-based data analysis workflow. This method enabled the identification of metal-binding compounds in complex samples based on defined mass (m/z) offsets of ion species with the same chromatographic profiles. As this native electrospray metabolomics approach is suited to the use of any liquid chromatography-mass spectrometry system to explore the binding of any metal, this method has the potential to become an essential strategy for elucidating metal-binding molecules in biology.

Recent strategies used in the synthesis of saturated four-membered heterocycles.

The importance and prevalance of O-, N-, and S-atom containing saturated four-membered ring motifs in biologically active molecules and potential therapeutics continues to drive efforts in their efficient synthetic preparation. In this review, general and recent strategies for the synthesis of these heterocycles are presented. Due to the limited potential bond disconnections, retrosynthetic strategies are broadly limited to cyclizations and cycloadditions. Nonetheless, diverse approaches for accessing cyclization precursors have been developed, ranging from nucleophilic substitution to C-H functionalization. Innovative methods for substrate activation have been developed for cycloadditions under photochemical and thermal conditions. Advances in accessing oxetanes, azetidines, and thietanes remain active areas of research with continued breakthroughs anticipated to enable future applications.