Elucidation of the crystal structure of the clorothricin decalin synthase of Streptomyces antibioticus Monserrat Manzo Ruiz, Catherine R. Back, Nicholas J. Burton, Paul R. Race University of Bristol, UK Introduction: Streptomyces antibioticus produces the spirotetonate antibiotic chlorothricin 1 . The biosynthesis of this natural product involves two cyclisation reactions, one of which is catalyzed by the enzyme ChlE3 and forms the distinctive trans -decalin ring of this compound 2 . To understand this reaction the crystal structure of ChlE3 has been determined. Methods: ChlE3 was cloned into pET29b. E. coli BL21 (DE3) was used for protein expression, 1 mM IPTG was added for induction, incubating at 20°C for 16 hrs. ChlE3 was purified through nickel column and further size exclusion chromatography. Crystallisation trials were established using the sitting drop method. Results and conclusions: Multiple large yellow crystals appeared in a range of different conditions, the best diffraction data was obtained from a crystal grown in lithium chloride, PEG and Tris-HCl. Diffraction data were collected at Diamond Light Source using beamline I24. The structure of ChlE3 was determined by molecular replacement and refined to 1.92 Å resolution. An overall completeness of 99.9% was achieved with a multiplicity of 6.5. Inspection of the ChlE3 structure reveals the presence of an active site bound flavin adenine dinucleotide (FAD) cofactor and implies a mechanism for intramolecular [4+2] cycloaddition in this biocatalyst. References 1. Jia, X. Y.; Tian, Z. H.; Shao, L.; Qu, X. D.; Zhao, Q. F.; Tang, J.; Tang, G. L.; Liu, W. Genetic characterization of the chlorothricin gene cluster as a model for spirotetronate antibiotic biosynthesis. Chem Biol. 2006, 13 (6), 575-85. DOI:10.1016/j.chembiol.2006.03.008 2. Tian, Z.; Sun, P.; Yan, Y.; Wu, Z.; Zheng, Q.; Zhou, S.; Zhang, H.; Yu, F.; Jia, X.; Chen, D.; Mándi, A.; Kurtán, T.; Liu, W. An enzymatic [4+2] cyclization cascade creates the pentacyclic core of pyrroindomycins. Nat Chem Biol. 2015, 11 (4), 259-65. DOI:10.1038/nchembio.1769
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