Abstract
Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged.
Highlights
Thiyl protein-based radical intermediates (Fig. 1), which is channeled into anaerobic metabolism [1, 11, 12]
We proposed that cleaved Pyruvate formate-lyase (PFL) (cPFL), which is cleaved at the glycyl radical at position 734, must be further truncated to truncated PFL construct (tPFL) to allow the C-terminal structured domain of YfiD to bind (Fig. 3A, iii)
We have further proposed that glycyl radical installation on YfiD by PFL-activating enzyme (AE) occurs after YfiD binds to cPFL (Fig. 3A, iv to v) [28], which would prevent glycyl radical quenching prior to YfiD association with cPFL and would protect the cell from unwanted radical chemistry
Summary
Thiyl protein-based radical intermediates (Fig. 1), which is channeled into anaerobic metabolism [1, 11, 12]. We proposed that cPFL, which is cleaved at the glycyl radical at position 734, must be further truncated to tPFL (residues 696–733 removed) to allow the C-terminal structured domain of YfiD to bind (Fig. 3A, iii).
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