Repairing enzymes using spare parts

Abstract

Glycyl radical enzymes (GREs) employ a post‐translationally installed glycyl radical species to carry out a wide range of chemistry in bacteria. The glycyl radical cofactor is a simple and effective catalyst, but it has an Achilles’ heel – it is susceptible to cleavage by molecular oxygen. For this reason, GREs are commonly expressed in bacteria under anoxic conditions, but not exclusively. The most well studied GRE, pyruvate formate lyase (PFL), is constitutively expressed, and this enzyme acts in primary metabolism, converting pyruvate and coenzyme A into acetyl‐CoA and formate, and is thus not an expendable enzyme. In this presentation, a mechanism for PFL repair from oxidative damage will be presented. In particular, we will consider how a small (14 kDa) ‘spare part protein’ termed YfiD can bind to an oxygen‐damaged PFL (PFL cleaved at Gly734) and restore activity. Our mechanism for repair takes advantage of multiple pieces of data from multiple biochemical and biophysical methods. Through making numerous constructs of both YfiD and PFL, we were able to use enzymology and spectroscopy to determine the regions of each protein that are required for YfiD‐PFL complex formation and activity restoration. We further were able to probe the molecular basis for glycyl radical installation on YfiD and the molecular signal that indicates that PFL is damaged and needs repair but is not too damaged for successful repair. Typically, damaged enzymes are cleared from the cell and new enzymes are made. Here, through use of a spare part protein, the 170‐kDa PFL gets a second chance.