Property optimization and substrate selectivity of a polyketide chain-releasing subunit CalA3 in calcimycin synthesis
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    Abstract:

    [Objective] CalA3 subunit catalyzes the release of the polyketide chain in the biosynthesis of calcimycin. This study aims to obtain CalA3 proteins with stable biochemical properties and homogeneous structure for cryo-electron microscopy, to understand the mechanism of polyketide chain releasing by the module of the polyketide synthase, and to explore the selectivity of CalA3 to polyketide substrates with different structures, thereby providing biochemical materials for the complexes of CalA3 with small ligands and a reference for further exploring the catalytic potential of CalA3. [Methods] The medium for culturing the heterologous expression strain and the biochemical conditions for CalA3 protein purification were optimized. Then, the CalA3 proteins were analyzed by negative-stain transmission electron microscopy. The reaction products of S-(2-acetamidoethyl)3-(1H-pyrrol-2-yl) propanethioate (SANC-C3), N-(2-(pentylthio) ethyl) acetamide (SNAC-C5), lauroyl-CoA and 3-hydroxy anthranilic acid (3HA) catalyzed by CalA3 were identified by high performance liquid chromatography and high-resolution mass spectrometry (LC-MS). [Results] The strains cultured in the optimized medium PGTY, expressed more CalA3 proteins which exhibited featured structures under negative-stain transmission electron microscope. The purified CalA3 proteins were prepared for cryo-electron microscopy for structural determination. CalA3 catalyzed the aminolysis reaction of SNAC-C5 and SNAC-C3 with 3HA. However, the products of lauroyl-CoA and 3HA catalyzed by CalA3 were not detected. [Conclusion] The results of protein purification and negative-stain transmission electron micrographs show that the conditions for culturing Escherichia coli for heterologous expression and purifying CalA3 proteins are established. The results of in vitro catalytic experiments demonstrate that CalA3 has broad selectivity for the structure of polyketide substrates. These findings provide a reference for further exploring the structure, functions, and application of polyketide synthases.

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LI Xixi, WANG Jialiang, CAO Wei, LI Dandan, DENG Zixin, WANG Zhijun, LIANG Jingdan. Property optimization and substrate selectivity of a polyketide chain-releasing subunit CalA3 in calcimycin synthesis. [J]. Acta Microbiologica Sinica, 2023, 63(1): 194-205

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History
  • Received:April 14,2022
  • Revised:May 15,2022
  • Online: January 13,2023
  • Published: January 04,2023
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