Anaerobic 4‐Hydroxyproline Metabolism by a Widespread Microbial Glycyl Radical Enzyme
Authors: Huang Yolanda Y. , Backman Lindsey , Gold Brian , Raines Ronald T. , Drennan Catherine L. , Balskus Emily P. 
Published: June 2021

Microbiome research has rapidly advanced through the use of high‐through put sequencing technologies, but the functions of most genes in sequence databases remain unknown. Glycyl radical enzymes represent an ancient protein super family, which is one of the most abundant protein families in the human gut microbiome. These enzymes use radical chemistry to catalyze a diverse set of chemically difficult transformations in metabolic pathways essential for anaerobic growth.Here, we describe the biochemical characterization of a new member of the glycyl radical enzyme superfamily, 4‐hydroxyproline dehydratase (HypD). This enzyme is responsible for the first step of anaerobic 4‐hydroxyproline (Hyp) metabolism. Hyp is the most abundant post‐translationally modified amino acid in the human proteome, with collagen as its principle source. It is also abundant in plant cell wall glycoproteins and functions as a site for O‐glycosylation. Hyp is present at significant levels in the human gut, deriving from both dietary sources and endogenous collagen turnover. Anaerobic Hyp metabolism had only been observed in Clostridiales as a part of Stickland fermentation in energy metabolism, but the enzymes and intermediates involved had not been identified until our work.We predicted the catalytic activity of HypD based on its sequence similarity to characterized glycyl radical enzymes and its genomic context in Clostridial species. Using purified enzymes, we demonstrated that HypD catalyzes the removal of water from Hyp to form pyrroline‐5‐carboxylate, a central intermediate in amino acid metabolism. As with all other glycyl radical enzymes, HypD activity requires post‐translational modification by a partner radical SAM enzyme to install a radical on a conserved glycine in the active site. We next examined the substrate scope and kinetics of HypD and demonstrated that it is stereospecific for trans‐4‐hydroxy‐L‐proline. Additional biochemical work has yielded insights into the radical‐based mechanism of this intriguing transformation. Our discovery of the biochemical basis for anaerobic Hyp metabolism has enabled us to uncover hundreds of strains with the potential for metabolizing Hyp. This under appreciated metabolism is encoded in species not known to ferment amino acids, where Hyp is likely used as a precursor for non‐Stickland pathways. In addition, HypD is widespread in common gut commensals as well as pathogens like Clostridiodes difficile, suggesting that it may represent a core function in the human gut microbiota. Furthermore, our work on the characterization of HypD has led us to new research directions to investigate related pathways in the gut microbiome.Support or Funding InformationFunded by Harvard University, a Packard Fellowship for Science and Engineering, and NSERC Postgraduate Scholarship‐Doctoral Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.