Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

Katja Zorn; Catherine R. Back; Rob Barringer; Veronika Chadimová; Monserrat Manzo‐Ruiz; Sbusisiwe Z. Mbatha; Juan‐Carlos Mobarec; Sam E. Williams; Marc W. van der Kamp; Paul R. Race; Christine L. Willis; Martin A. Hayes

doi:10.1002/cbic.202300382

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

Katja Zorn, Catherine R. Back, Rob Barringer, Veronika Chadimová, Monserrat Manzo‐Ruiz, Sbusisiwe Z. Mbatha, Juan‐Carlos Mobarec, Sam E. Williams, Marc W. van der Kamp, Paul R. Race, Christine L. Willis, Martin A. Hayes

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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.

Original language	English
Article number	e202300382
Journal	ChemBioChem
Volume	24
Issue number	14
Early online date	12 Jun 2023
DOIs	https://doi.org/10.1002/cbic.202300382
Publication status	Published - 17 Jul 2023

Bibliographical note

Funding Information:
This study was supported by BBSRC grants BB/T001968/1, BB/M012107/1, BB/S507908/1 and BB/M025624/1, and through the award of Ph.D studentships to RB (BBSRC SWBio; BB/T008741/1), SZM (EPSRC CDT in Chemical Synthesis; EP/L015366/1), SEW (MRC GW4 BIOMED DTP; MR/N0137941/1) and MM‐R (Conacyt;766585), and by AstraZeneca. K.Z. was funded by the AstraZeneca postdoc programme. V.C. was sponsored by the AstraZeneca R&D Graduate programme. We thank Ileana Guzzetti, Giovanna Mejia, Pernilla Korsgren, Dusan Petrovic, and Paul Curnow for their technical and intellectual support, and the Diamond Light Source staff for assistance with X‐ray diffraction data collection.

Publisher Copyright:
© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.

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Zorn, K., Back, C. R., Barringer, R., Chadimová, V., Manzo‐Ruiz, M., Mbatha, S. Z., Mobarec, JC., Williams, S. E., Kamp, M. W. V. D., Race, P. R., Willis, C. L., & Hayes, M. A. (2023). Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases. ChemBioChem, 24(14), Article e202300382. https://doi.org/10.1002/cbic.202300382

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title = "Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases",

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.",

author = "Katja Zorn and Back, {Catherine R.} and Rob Barringer and Veronika Chadimov{\'a} and Monserrat Manzo‐Ruiz and Mbatha, {Sbusisiwe Z.} and Juan‐Carlos Mobarec and Williams, {Sam E.} and Kamp, {Marc W. van der} and Race, {Paul R.} and Willis, {Christine L.} and Hayes, {Martin A.}",

note = "Funding Information: This study was supported by BBSRC grants BB/T001968/1, BB/M012107/1, BB/S507908/1 and BB/M025624/1, and through the award of Ph.D studentships to RB (BBSRC SWBio; BB/T008741/1), SZM (EPSRC CDT in Chemical Synthesis; EP/L015366/1), SEW (MRC GW4 BIOMED DTP; MR/N0137941/1) and MM‐R (Conacyt;766585), and by AstraZeneca. K.Z. was funded by the AstraZeneca postdoc programme. V.C. was sponsored by the AstraZeneca R&D Graduate programme. We thank Ileana Guzzetti, Giovanna Mejia, Pernilla Korsgren, Dusan Petrovic, and Paul Curnow for their technical and intellectual support, and the Diamond Light Source staff for assistance with X‐ray diffraction data collection. Publisher Copyright: {\textcopyright} 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.",

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doi = "10.1002/cbic.202300382",

language = "English",

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TY - JOUR

T1 - Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

AU - Zorn, Katja

AU - Back, Catherine R.

AU - Barringer, Rob

AU - Chadimová, Veronika

AU - Manzo‐Ruiz, Monserrat

AU - Mbatha, Sbusisiwe Z.

AU - Mobarec, Juan‐Carlos

AU - Williams, Sam E.

AU - Kamp, Marc W. van der

AU - Race, Paul R.

AU - Willis, Christine L.

AU - Hayes, Martin A.

N1 - Funding Information: This study was supported by BBSRC grants BB/T001968/1, BB/M012107/1, BB/S507908/1 and BB/M025624/1, and through the award of Ph.D studentships to RB (BBSRC SWBio; BB/T008741/1), SZM (EPSRC CDT in Chemical Synthesis; EP/L015366/1), SEW (MRC GW4 BIOMED DTP; MR/N0137941/1) and MM‐R (Conacyt;766585), and by AstraZeneca. K.Z. was funded by the AstraZeneca postdoc programme. V.C. was sponsored by the AstraZeneca R&D Graduate programme. We thank Ileana Guzzetti, Giovanna Mejia, Pernilla Korsgren, Dusan Petrovic, and Paul Curnow for their technical and intellectual support, and the Diamond Light Source staff for assistance with X‐ray diffraction data collection. Publisher Copyright: © 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.

PY - 2023/7/17

Y1 - 2023/7/17

N2 - 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.

AB - 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.

UR - https://doi.org/10.1002/cbic.202300382

U2 - 10.1002/cbic.202300382

DO - 10.1002/cbic.202300382

M3 - Article (Academic Journal)

C2 - 37305956

SN - 1439-4227

VL - 24

JO - ChemBioChem

JF - ChemBioChem

IS - 14

M1 - e202300382

ER -

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

Abstract

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