6MLK

Structure of Thioesterase from DEBS with a thioesterase-specific antibody

Summary for 6MLK

• Entry DOI: 10.2210/pdb6mlk/pdb
• Descriptor: 6-deoxyerythronolide-B synthase EryA3, modules 5 and 6, Light chain of Fab 3A6, Heavy chain of Fab 3A6, ... (5 entities in total)
• Functional Keywords: 6-deoxyerythronolide b synthase, thioesterase, monoclonal antibody, transferase
• Biological source: Saccharopolyspora erythraea; More
• Total number of polymer chains: 3
• Total formula weight: 84440.60

Authors

Mathews, I.I.,Li, X.,Khosla, C. (deposition date: 2018-09-27, release date: 2018-10-17, Last modification date: 2024-10-16)

Primary citation

Li, X.,Sevillano, N.,La Greca, F.,Hsu, J.,Mathews, I.I.,Matsui, T.,Craik, C.S.,Khosla, C.
Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase.
Biochemistry, 57:6201-6208, 2018

PubMed Abstract: Assembly line polyketide synthases (PKSs) are large multimodular enzymes responsible for the biosynthesis of diverse antibiotics in bacteria. Structural and mechanistic analysis of these megasynthases can benefit from the discovery of reagents that recognize individual domains or linkers in a site-specific manner. Monoclonal antibodies not only have proven themselves as premier tools in analogous applications but also have the added benefit of constraining the conformational flexibility of their targets in unpredictable but often useful ways. Here we have exploited a library based on the naïve human antibody repertoire to discover a F (3A6) that recognizes the terminal thioesterase (TE) domain of the 6-deoxyerythronolide B synthase with high specificity. Biochemical assays were used to verify that 3A6 binding does not inhibit enzyme turnover. The co-crystal structure of the TE-3A6 complex was determined at 2.45 Å resolution, resulting in atomic characterization of this protein-protein recognition mechanism. F binding had minimal effects on the structural integrity of the TE. In turn, these insights were used to interrogate via small-angle X-ray scattering the solution-phase conformation of 3A6 complexed to a catalytically competent PKS module and bimodule. Altogether, we have developed a high-affinity monoclonal antibody tool that recognizes the TE domain of the 6-deoxyerythronolide B synthase while maintaining its native function.

PubMed: 30289692
DOI: 10.1021/acs.biochem.8b00886

Experimental method

X-RAY DIFFRACTION (2.45 Å)