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Interrogation of an enzyme library reveals the catalytic plasticity of naturally evolved [4+2] cyclases

DOI: 10.1002/cbic.202300382 DOI Help

Authors: Katja Zorn (AstraZeneca Sweden) , Catherine R. Back (University of Bristol) , Rob Barringer (University of Bristol) , Veronika Chadimová (AstraZeneca Sweden) , Monserrat Manzo-Ruiz (University of Bristol) , Sbusisiwe Z. Mbatha (University of Bristol) , Juan-Carlos Mobarec (AstraZeneca) , Sam E. Williams (University of Bristol) , Marc W. Van Der Kamp (University of Bristol) , Paul R. Race (University of Bristol) , Christine L. Willis (University of Bristol) , Martin Alistair Hayes (AstraZeneca Sweden)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Chembiochem

State: Published (Approved)
Published: June 2023
Diamond Proposal Number(s): 23269

Open Access Open Access

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.

Journal Keywords: cyclases; biosynthetic gene cluster; polyketides; enzyme screening; intra-molecular Diels-Alder reaction

Diamond Keywords: Enzymes

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography

Added On: 14/06/2023 10:16

Documents:
ChemBioChem - 2023 - Zorn - Interrogation of an enzyme library reveals the catalytic plasticity of naturally evolved 4 2 .pdf

Discipline Tags:

Biochemistry Chemistry Structural biology Organic Chemistry Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)