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.
Support or Funding Information
This work was supported in part by National Institutes of Health (NIH) grants R35 GM126982 (CLD), F32 GM129882 (MCA), F32 GM133056 (ECU), and P30-ES002109 (core grant from NIEHS). CLD is a Howard Hughes Medical Institute (HHMI) Investigator. LRFB is a recipient of a Dow Fellowship at MIT, a National Science Foundation (NSF) Graduate Research Fellowship under Grant No. 1122374 (LRFB), and a Gilliam Fellowship from HHMI. PLL was funded by the MIT UROP office.
lt;pgt;This work was supported in part by National Institutes of Health (NIH) grants R35 GM126982 (CLD), F32 GM129882 (MCA), F32 GM133056amp;nbsp;(ECU), and P30-ES002109 (core grant from NIEHS). CLD is a Howard Hughes Medical Institute (HHMI) Investigator. LRFB is a recipient of a Dow Fellowship at MIT, a National Science Foundation (NSF) Graduate Research Fellowship under Grant No. 1122374 (LRFB), and a Gilliam Fellowship from HHMI. PLL was funded by the MIT UROP office.lt;/pgt; lt;stylegt;@font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-469750017 -1073732485 9 0 511 0;}@font-face {font-family:ArialMT; panose-1:2 11 6 4 2 2 2 2 2 4; mso-font-alt:Arial; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:auto; mso-font-signature:0 0 0 0 0 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}div.WordSection1 {page:WordSectionlt;/stylegt;
