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J Biol Inorg Chem. 2019 Jun 27. doi: 10.1007/s00775-019-01681-2. [Epub ahead of print]

Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen-damaged glycyl radical enzyme.

Author information

1
Howard Hughes Medical Institute, Massachusetts Institute of Technology, Building 68-680, Cambridge, MA, 02139, USA.
2
Department of Chemistry, Massachusetts Institute of Technology, Building 68-680, Cambridge, MA, 02139, USA.
3
Hauptman-Woodward Medical Research Institute, Buffalo, NY, 14203, USA.
4
MIT Summer Research Program (MSRP), Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
5
Department of Biology, Massachusetts Institute of Technology, Building 68-680, Cambridge, MA, 02139, USA.
6
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, 94720, USA.
7
Department of Chemistry, Princeton University, Princeton, NJ, 08544, USA.
8
Electrical Engineering and Computer Science and Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, 02319, USA.
9
Division of Cardiovascular Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA.
10
Howard Hughes Medical Institute, Massachusetts Institute of Technology, Building 68-680, Cambridge, MA, 02139, USA. cdrennan@mit.edu.
11
Department of Chemistry, Massachusetts Institute of Technology, Building 68-680, Cambridge, MA, 02139, USA. cdrennan@mit.edu.
12
Department of Biology, Massachusetts Institute of Technology, Building 68-680, Cambridge, MA, 02139, USA. cdrennan@mit.edu.
13
Center for Environmental Health, Massachusetts Institute of Technology, Building 68-680, Cambridge, MA, 02139, USA. cdrennan@mit.edu.

Abstract

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.

KEYWORDS:

Circular dichroism; Cofactor repair; Glycyl radical enzyme; Nuclear magnetic resonance; Radical chemistry

PMID:
31250200
DOI:
10.1007/s00775-019-01681-2

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