Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases |
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Authors: | Dr Katja Zorn Dr Catherine R Back Rob Barringer Veronika Chadimová Monserrat Manzo-Ruiz Dr Sbusisiwe Z Mbatha Juan-Carlos Mobarec Dr Sam E Williams Dr Marc W van der Kamp Prof Paul R Race Prof Christine L Willis Prof Martin A Hayes |
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Affiliation: | 1. Compound Synthesis and Management, Discovery Sciences, Biopharmaceuticals R&D, AstraZeneca, Pepparedsleden 1, 431 83 Mölndal, Sweden;2. School of Biochemistry, University of Bristol, Bristol, BS8 1TD UK;3. School of Chemistry, University of Bristol, Bristol, BS8 1TS UK;4. Mechanistic and Structural Biology, Biopharmaceuticals R&D, AstraZeneca, Cambridge, CB21 6GH UK |
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Abstract: | Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a 4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved 4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted 4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases. |
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Keywords: | cyclases biosynthetic gene cluster polyketides enzyme screening intra-molecular Diels-Alder reaction |
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