Assay Development to Probe Enzymatic Heterocyclization by Fluorescence

• DOI: 10.1096/fasebj.2018.32.1_supplement.526.16
• Publication Date: 2021-06-21T16:23:16Z
• Authors: Mark MacRae; Viet Dieu; MacKenzie Patterson; Bryan Henriquez; Jessica Soule; Yuan Xia; Daniel P. Dowling
• Keywords: 0303 health sciences; 03 medical and health sciences

Many therapeutically useful natural products contain 5‐membered heterocycles derived from either serine, cysteine or threonine, and important examples include the anticancer epothilones and bleomycin. In nonribosomal peptide synthetase systems, formation of these rings is catalyzed by a specific functional domain, the cyclization (Cy) domain, which catalyzes both a bond‐forming condensation reaction and a subsequent cyclodehydration reaction. To probe the reaction mechanism of this system, we are exploring the Cy domain from the protein EpoB from Sorangium cellulosum as a model system. The EpoB Cy structure has been previously characterized and determined to contain two active site channels acting as binding sites for substrate T domains. Here, we report the development of a fluorescence plate‐reader assay to monitor the cyclization reaction. Using this assay, we are exploring the functional role of residues within the Cy domain active site.Support or Funding InformationNational Institute of General Medical Sciences of the National Institutes of Health Award Number 1R15GM123425‐01 to D.P.D.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.