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Interrogation of an enzyme library reveals the catalytic plasticity of naturally evolved [4+2] cyclases
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
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
Discipline Tags:
Biochemistry
Chemistry
Structural biology
Organic Chemistry
Life Sciences & Biotech
Technical Tags:
Diffraction
Macromolecular Crystallography (MX)