Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase

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 Fab (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. Fab 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.

Antibody Probes of Module 1 of the 6-Deoxyerythronolide B Synthase Reveal an Extended Conformation During Ketoreduction

“…In both cases, cocrystal structures were obtained of the F ab and domain partner complexes, affording insights into the molecular details of antibody recognition. 20,21 The structure of 1B2 in complex with the KS-AT didomain from M3 recapitulated the previously observed extended conformation. 22−24 Kinetic analyses measured comparable k cat and K M values of M3 in the presence and absence of 1B2, suggesting the extended conformation is catalytically competent for the essential rate-limiting reactions (i.e., entry translocation and elongation).…”

“…Antibodies 1B2 and 3A6 bind with high-affinity to the KS-AT didomain of DEBS M3 and the TE domain of DEBS M6, respectively (Figure A). In both cases, cocrystal structures were obtained of the F ab and domain partner complexes, affording insights into the molecular details of antibody recognition. , The structure of 1B2 in complex with the KS-AT didomain from M3 recapitulated the previously observed extended conformation. − Kinetic analyses measured comparable k cat and K M values of M3 in the presence and absence of 1B2, suggesting the extended conformation is catalytically competent for the essential rate-limiting reactions (i.e., entry translocation and elongation). Further support for the catalytic relevance of the extended model came from SEC-SAXS analysis of 1B2-bound M3 in distinct catalytic states …”

Thioesterase enzyme families: Functions, structures, and mechanisms

“…TE17 has enzymes, which are the TE domain of macrocycle-forming PKSs, such as of 6-deoxyerythronolide B synthase from S. erythraea, 135,136,172,173 picromycin synthase from S. venezuelae, 136,174,175 and tautomycin synthase. 137 They all show a consistent Ser-Asp-His catalytic triad.…”

Fragment antigen binding domains (Fabs) as tools to study assembly-line polyketide synthases

“…One portion of the PKS that remains especially difficult to model is the flexible TE domain [ 61 ]. The structure of a F ab that recognizes the DEBS TE (3A6, PDB ID 6MLK ) has been reported; this can be considered a neutral F ab because the TE maintains its catalytic activity as part of the complex [ 61 , 63 ]. As previously reported ( Fig.…”