Identification of a Novel Cobamide Remodeling Enzyme in the Beneficial Human Gut Bacterium Akkermansia muciniphila

Abstract

The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain MucT is known to use cobamides, the vitamin B12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila MucT is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila MucT were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling—the removal and replacement of the lower ligand—despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila. We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli. Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment. IMPORTANCE Cobamides, comprising the vitamin B12 family of cobalt-containing cofactors, are required for metabolism in all domains of life, including most bacteria. Cobamides have structural variability in the lower ligand, and selectivity for particular cobamides has been observed in most organisms studied to date. Here, we discovered that the beneficial human gut bacterium Akkermansia muciniphila can use a diverse range of cobamides due to its ability to change the cobamide structure via a process termed cobamide remodeling. We identify and characterize the novel enzyme CbiR that is necessary for initiating the cobamide remodeling process. The discovery of this enzyme has implications for understanding the ecological role of A. muciniphila in the gut and the functions of other bacteria that produce this enzyme.

The Protective Effects of Live and Pasteurized Akkermansia muciniphila and Its Extracellular Vesicles against HFD/CCl4-Induced Liver Injury

“…In addition, A. muciniphila is known as one of the few bacterial species capable of using mucin as a sole energy source and releases products that are used by butyrate-producing bacteria. Because of this mucin-degrading ability, it is believed that this bacterium is a keystone species in the healthy gut ( 60 ). Furthermore, a recent study shows that live A. muciniphila had major effects on metabolism in the gut and the liver, while the pasteurized A. muciniphila was more effective in elevating the beneficial metabolites, especially in the gut of antibiotic treated mice ( 61 ).…”

A roadmap for metagenomic enzyme discovery

“…For example, a new family of cobamide-remodeling enzymes widespread in the human gut microbiome was identified based on genome context analysis of a coding sequence of unknown function flanked by cobamide biosynthesis and salvaging genes. 139 To automate genome neighborhood analysis, a widely used addition to the EFI-EST is the Genome Neighborhood Tool (GNT). 140 EFI-GNT generates genome neighborhood networks and allows for rapid visual assessment of genome context.…”

Cobalamin riboswitches are broadly sensitive to corrinoid cofactors to enable an efficient gene regulatory strategy

“…To measure concentrations of corrinoid stock solutions, corrinoid samples were diluted 10-fold in 10 mM sodium cyanide to obtain the dicyanated base-off form of the corrinoid. The concentration of the dicyanated corrinoid was calculated using the extinction coefficient ε580 = 10.1 mM −1 cm −1 (Mok et al, 2020; Stupperich et al, 1988). For adenosylated corrinoids used in Figures 4 and S7, base-on/off constitution at neutral pH was measured as the ratio of spectral absorbance at 525 nm and 458 nm in phosphate buffered saline solution pH 7.3 at 37°C (Fieber et al, 2002; Hoffmann et al, 2000).…”