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  • Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase. Biochemistry Guzman, K. M., Cogan, D. P., Brodsky, K. L., Soohoo, A. M., Li, X., Sevillano, N., Mathews, I. I., Nguyen, K. P., Craik, C. S., Khosla, C. 2023


    Fragment antigen-binding domains of antibodies (Fabs) are powerful probes of structure-function relationships of assembly line polyketide synthases (PKSs). We report the discovery and characterization of Fabs interrogating the structure and function of the ketosynthase-acyltransferase (KS-AT) core of Module 2 of the 6-deoxyerythronolide B synthase (DEBS). Two Fabs (AC2 and BB1) were identified to potently inhibit the catalytic activity of Module 2. Both AC2 and BB1 were found to modulate ACP-mediated reactions catalyzed by this module, albeit by distinct mechanisms. AC2 primarily affects the rate (kcat), whereas BB1 increases the KM of an ACP-mediated reaction. A third Fab, AA5, binds to the KS-AT fragment of DEBS Module 2 without altering either parameter; it is phenotypically reminiscent of a previously characterized Fab, 1B2, shown to principally recognize the N-terminal helical docking domain of DEBS Module 3. Crystal structures of AA5 and 1B2 bound to the KS-AT fragment of Module 2 were solved to 2.70 and 2.65 A resolution, respectively, and revealed entirely distinct recognition features of the two antibodies. The new tools and insights reported here pave the way toward advancing our understanding of the structure-function relationships of DEBS Module 2, arguably the most well-studied module of an assembly line PKS.

    View details for DOI 10.1021/acs.biochem.3c00156

    View details for PubMedID 37184546