Lindsey Backman (Massachusetts Institute of Technology), Yolanda Huang (Lawrence Berkeley National Laboratory), Mary Andorfer (Massachusetts Institute of Technology), Brian Gold (University of New Mexico), Ronald Raines (Massachusetts Institute of Technology), Emily Balskus (Harvard University, Howard Hughes Medical Institute), Catherine Drennan (Massachusetts Institute of Technology, Howard Hughes Medical Institute)
Presenting Author Massachusetts Institute of Technology
The glycyl radical enzyme (GRE) family utilizes a glycyl radical cofactor, installed by AdoMet radical activating enzymes, to catalyze difficult chemical reactions in a variety of microbial metabolic pathways. Although GREs are widely encoded and expressed by bacteria found in the gut microbiome, these enzymes remain largely uncharacterized. Recently a new GRE, hydroxyproline dehydratase (HypD) was discovered to catalyze the dehydration of trans-4-hydroxy-L-proline (4-Hyp) to 1-pyrroline-5-carboxylic acid. HypD is the second most prominent GRE in the human gut microbiome and is encoded by 360 bacterial genomes, including the commonly antibiotic-resistant human pathogen C. difficile. HypD presents a pathway for bacteria to reverse 4-Hyp post-translational modifications, the most common post-translational modification in animals, which was previously thought to be irreversible. Furthermore, the bacteria that encode HypD are known to use 4-Hyp as an electron acceptor during amino acid fermentation, their primary method of generating adenosine triphosphate (ATP). However, the enzyme responsible for assimilating 4-Hyp into this pathway has remained unknown until now. HypD could be the missing puzzle piece to understanding how these bacteria use the abundant metabolite 4-Hyp in energy production, while also symbiotically providing humans with a method for recycling this common amino acid. In order to elucidate the mechanism for how HypD performs the dehydration of hydroxyproline, we aimed to characterize HypD from C. difficile, in the presence of its substrate. Here, we have solved a 2.05-Å resolution structure for HypD by molecular replacement. Subsequently, a structure for HypD with its substrate 4-Hyp bound in the active site was solved to 2.52-Å resolution. These structures, accompanied by site-directed mutagenesis studies and biochemical assays with deuterated substrate, have led us to identify key catalytic residues and have provided insight into the radical mechanism for 4-Hyp dehydration.
Support or Funding Information
This work was supported in part by National Institutes of Health (NIH) Grant Nos. R01 GM069857 (CLD), R35 GM126982 (CLD), F32 GM129882 (MCA), and R01 GM044783 (RTR); the National Science Foundation (NSF) Graduate Research Fellowship under Grant Nos. 1122374 (LRFB); Harvard University (EPB); a Packard Fellowship for Science and Engineering (2013-39267) (EPB); the NSERC Postgraduate Scholarship-Doctoral Program (YYH); and an Arnold O. Beckman Postdoctoral Fellowship (BG). CLD and EPB. are Howard Hughes Medical Institute (HHMI) Investigators. LRFB is a recipient of a Dow Fellowship at MIT and a Gilliam Fellowship from HHMI. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357 (Beamline 24-IDC). This work was completed in part with resources at the MIT Center for Environmental Health Sciences and the MIT Dept. of Chemistry Instrumentation Facility.
lt;pgt;This work was supported in part by National Institutes of Health (NIH) Grant Nos. R01 GM069857 (CLD), R35 GM126982 (CLD), F32 GM129882 (MCA), and R01 GM044783 (RTR); the National Science Foundation (NSF) Graduate Research Fellowship under Grant Nos. 1122374 (LRFB); Harvard University (EPB); a Packard Fellowship for Science and Engineering (2013-39267) (EPB); the NSERC Postgraduate Scholarship-Doctoral Program (YYH); and an Arnold O. Beckman Postdoctoral Fellowship (BG). CLD and EPB. are Howard Hughes Medical Institute (HHMI) Investigators. LRFB is a recipient of a Dow Fellowship at MIT and a Gilliam Fellowship from HHMI. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357 (Beamline 24-IDC). This work was completed in part with resources at the MIT Center for Environmental Health Sciences and the MIT Dept. of Chemistry Instrumentation Facility.lt;/pgt;
Structural and biochemical characterization of the newly discovered glycyl radical enzyme HypD (PDB ID: 6VXE) from the pathogen C. difficile has provided insight into this radical-based enzyme mechanism. This chemical understanding into HypD will allow for structure-based design of inhibitors for this promising drug target.
