Glycyl radical enzymes (GREs) utilize a glycyl radical cofactor to carry out a diverse array of chemically challenging enzymatic reactions in anaerobic bacteria. Although the glycyl radical is a powerful catalyst, it is also oxygen sensitive such that oxygen exposure causes cleavage of the GRE at the site of the radical. This oxygen sensitivity presents a challenge to facultative anaerobes dwelling in areas prone to oxygen exposure. Once GREs are irreversibly oxygen damaged, cells either need to make new GREs or somehow repair the damaged one. One particular GRE, pyruvate formate lyase (PFL), can be repaired through the binding of a 14.3 kDa protein, termed YfiD, which is constitutively expressed in E. coli. Herein, we have solved a solution structure of this ‘spare part’ protein using nuclear magnetic resonance spectroscopy. These data, coupled with data from circular dichroism, indicate that YfiD has an inherently flexible N-terminal region (residues 1–60) that is followed by a C-terminal region (residues 72–127) that has high similarity to the glycyl radical domain of PFL. Reconstitution of PFL activity requires that YfiD binds within the core of the PFL barrel fold; however, modeling suggests that oxygen-damaged, i.e. cleaved, PFL cannot fully accommodate YfiD. We further report that a PFL variant that mimics the oxygen-damaged enzyme is highly susceptible to proteolysis, yielding additionally truncated forms of PFL. One such PFL variant of ~ 77 kDa makes an ideal scaffold for the accommodation of YfiD. A molecular model for the rescue of PFL activity by YfiD is presented.
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Cleaved pyruvate formate lyase
Autonomous glycyl radical cofactor
Glycyl radical domain
Glycyl radical enzyme
Heteronuclear single quantum coherence
Immobilized metal affinity chromatography
Nuclear magnetic resonance
Nuclear Overhauser effect
Pyruvate formate lyase
Pyruvate formate lyase activating enzyme
PFL truncation product 1 at 77 kDa
PFL truncation product 2 at 71 kDa
Truncated YfiD with 60 N-terminal residues removed
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This work was supported in part by the National Institutes of Health (NIH) GM069857 (C.L.D.), R35 GM126982 (C.L.D.), F32 GM129882 (M.C.A.), and F32 GM099257 (S.E.J.B.), Analog Devices (C.M.S.), MIT-IBM Watson Lab (C.M.S.), MIT J-Clinic (C.M.S.), and the National Science Foundation (NSF) Graduate Research Fellowship under Grant No. 1122374 (L.R.F.B.). C.L.D is a Howard Hughes Medical Institute (HHMI) Investigator. S.Y. and L.R.F.B. were part of the HHMI EXROP program and the Bio-MIT Summer Research Program (MSRP) program. A.C. was also part of the MSRP program. L.R.F.B. is a recipient of a Dow Fellowship at MIT and a Gilliam Fellowship from HHMI. The Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems (NSF-0070319) is gratefully acknowledged. We would like to thank University of Connecticut Health Center NMR facility and Massachusetts Institute of Technology’s Francis Bitter Magnet Lab for instrument use. We acknowledge Miranda Lynch for helping with R scripting, and Laurel Kinman for helping with editing.
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Bowman, S.E.J., Backman, L.R.F., Bjork, R.E. et al. Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen-damaged glycyl radical enzyme. J Biol Inorg Chem 24, 817–829 (2019). https://doi.org/10.1007/s00775-019-01681-2
- Radical chemistry
- Glycyl radical enzyme
- Nuclear magnetic resonance
- Cofactor repair
- Circular dichroism