The genus of Parasutterella has been defined as a core component of the human and mouse gut microbiota, and has been correlated with various health outcomes. However, like most core microbes in the gastrointestinal tract (GIT), very little is known about the biology of Parasutterella and its role in intestinal ecology. In this study, Parasutterella was isolated from the mouse GIT and characterized in vitro and in vivo. Mouse, rat, and human Parasutterella isolates were all asaccharolytic and producers of succinate. The murine isolate stably colonized the mouse GIT without shifting bacterial composition. Notable changes in microbial-derived metabolites were aromatic amino acid, bilirubin, purine, and bile acid derivatives. The impacted bile acid profile was consistent with altered expression of ileal bile acid transporter genes and hepatic bile acid synthesis genes, supporting the potential role of Parasutterella in bile acid maintenance and cholesterol metabolism. The successful colonization of Parasutterella with a single environmental exposure to conventional adult mice demonstrates that it fills the ecological niche in the GIT and contributes to metabolic functionalities. This experiment provides the first indication of the role of Parasutterella in the GIT, beyond correlation, and provides insight into how it may contribute to host health.

SummaryAcireductone dioxygenase (ARD) catalyzes different reactions between O 2 and 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (acireductone) depending upon the metal bound in the active site. Ni +2 -ARD cleaves acireductone to formate, CO and methylthiopropionate. If Fe +2 is bound (ARD ′), the same substrates yield methylthioketobutyrate and formate. The two forms differ in structure, and are chromatographically separable. Paramagnetism of Fe +2 renders the active site of ARD′ inaccessible to standard NMR methods. The structure of ARD′ has been determined using Fe +2 binding parameters determined by X-ray absorption spectroscopy and NMR restraints from H98S ARD, a metal-free diamagnetic protein that is isostructural with ARD′. ARD′ retains the β-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the β-sandwich and decreases order at the other upon interconversion of ARD and ARD′, causing loss of the C-terminal helix in ARD′ and rearrangements of residues involved in substrate orientation in the active site.

Here we report the structure of acireductone dioxygenase (ARD), the first determined for a new family of metalloenzymes. ARD represents a branch point in the methionine salvage pathway leading from methylthioadenosine to methionine and has been shown to catalyze different reactions depending on the type of metal ion bound in the active site. The solution structure of nickel-containing ARD (Ni-ARD) was determined using NMR methods. X-ray absorption spectroscopy, assignment of hyperfine shifted NMR resonances and conserved domain homology were used to model the metal-binding site because of the paramagnetism of the bound Ni2+. Although there is no structure in the Protein Data Bank within 3 A r.m.s deviation of that of Ni-ARD, the enzyme active site is located in a conserved double-stranded b-helix domain. Furthermore, the proposed Ni-ARD active site shows significant post-facto structural homology to the active sites of several metalloenzymes in the cupin superfamily.

Acireductone dioxygenases (ARDs) are enzymes involved in the methionine recycle pathway, which regulates aspects of the cell cycle. Klebsiella pneumoniae produces two enzymes that share a common polypeptide sequence and differ only in the metal ion present. Reaction of acireductone (1,2-dihydroxy-3-keto-5-methylthiopentene) with Fe-ARD and dioxygen produces formate and 2-keto-4-methylthiobutanoic acid, the alpha-ketoacid precursor of methionine. Ni-ARD reacts with acireductone and dioxygen to produce methylthiopropionate, CO, and formate and does not lie on the methionine recycle pathway. An X-ray absorption spectroscopy (XAS) study of the structure of the catalytic Ni center in resting Ni-ARD enzyme and the enzyme-substrate complex is reported. This study establishes the structure of the Ni site in resting Ni-ARD as containing a six coordinate Ni site composed of O/N-donor ligands including 3-4 histidine residues, demonstrates that the substrate binds to the Ni center in a bidentate fashion by displacing two ligands, at least one of which is a histidine ligand, and provides insight into the mechanism of catalysis employed by a Ni-containing dioxygenase. Efficiently relaxed and hyperfine-shifted resonances are observed in the (1)H nuclear magnetic resonance spectrum of Ni-ARD that can be attributed to the His imidazoles ligating the paramagnetic Ni ion and are consistent with the XAS results regarding His ligation. These resonances show significant perturbation in the presence of substrate, confirming that the metal ion interacts directly with the substrate.