A prominent glycyl radical enzyme in human gut microbiomes metabolizes trans -4-hydroxy- l -proline

Abstract

The human microbiome encodes vast numbers of uncharacterized enzymes, limiting our functional understanding of this community and its effects on host health and disease. By incorporating information about enzymatic chemistry into quantitative metagenomics, we determined the abundance and distribution of individual members of the glycyl radical enzyme superfamily among the microbiomes of healthy humans. We identified many uncharacterized family members, including a universally distributed enzyme that enables commensal gut microbes and human pathogens to dehydrate trans-4-hydroxy-L-proline, the product of the most abundant human posttranslational modification. This ‘chemically-guided functional profiling’ workflow can therefore use ecological context to facilitate the discovery of enzymes in microbial communities.

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity

“…5 We used this workflow to assess the distribution of GREs in Human Microbiome Project (HMP) metagenomes. Members of the GRE superfamily participate in anaerobic primary metabolism, including DNA synthesis (class III ribonucleotide reductase) and keto acid fermentation (pyruvate formate-lyase and ketobutyrate formate-lyase).…”

“…(A, D) Enhanced growth was observed for both C. difficile 630Δ erm and T. glycolicus DSM 1288 upon supplementation of a previously reported medium with either Pro or Hyp (20 mM final concentration). 5 Downstream metabolites were quantified at various timepoints in cultures supplemented with Pro (B, E) or Hyp (C, F). Experiments were set up as previously described 5 with the following modifications: 200 µL cultures were grown in 96-well plates and OD 600 measurements were taken using a PowerWave HT Microplate Spectrophotometer (BioTek).…”

“…5 Downstream metabolites were quantified at various timepoints in cultures supplemented with Pro (B, E) or Hyp (C, F). Experiments were set up as previously described 5 with the following modifications: 200 µL cultures were grown in 96-well plates and OD 600 measurements were taken using a PowerWave HT Microplate Spectrophotometer (BioTek). Samples were collected at various time points for metabolite quantification by liquid chromatography tandem-mass spectrometry.…”

“…Samples were collected at various time points for metabolite quantification by liquid chromatography tandem-mass spectrometry. 5 5-Aminovalerate was measured using the Hyp detection method by monitoring precursor and product ions of m/z 118.1 → m/z 101.1. Sample peak areas are normalized to the corresponding standard.…”

“…Previous BLAST searches revealed HypD in >850 genomes from the NCBI database, with the majority of hits found in Firmicutes and Bacteroidetes. 5 HypD is not just widely distributed across bacterial phyla but is also present in several archaeal genomes. Notably, we found HypD in many common oral and gut species from genera Treponema , Clostridium , Bacteroides , Parabacteroides , and Enterococcus (Fig.…”