TY - JOUR

T1 - Understanding and Engineering the Stereoselectivity of Humulene Synthase

AU - Schotte, Carsten

AU - Lukat, Peer

AU - Deuschmann, Adrian

AU - Blankenfeldt, Wulf

AU - Cox, Russell J.

N1 - Funding Information: CS thanks Leibniz University for funding. DFG is thanked for funding (INST 187/686‐1). Prof. Jeroen Dickschat is thanked for the gift of plasmid pYE‐BbS and Vanessa Harms is thanked for the gift of farnesyl pyrophosphate. Luca Codutti and Georg Krüger are thanked for MALS measurements. Ute Widow is thanked for technical assistance. We acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Parts of this research were carried out at beamline P11 at the PETRA III storage ring and we would like to thank the beamline staff for assistance during data collection. Beamtime was allocated for proposal Xh‐20010031. Open access funding enabled and organized by Projekt DEAL.

PY - 2021/8/30

Y1 - 2021/8/30

N2 - The non-canonical terpene cyclase AsR6 is responsible for the formation of 2E,6E,9E-humulene during the biosynthesis of the tropolone sesquiterpenoid (TS) xenovulene A. The structures of unliganded AsR6 and of AsR6 in complex with an in crystallo cyclized reaction product and thiolodiphosphate reveal a new farnesyl diphosphate binding motif that comprises a unique binuclear Mg 2+-cluster and an essential K289 residue that is conserved in all humulene synthases involved in TS formation. Structure-based site-directed mutagenesis of AsR6 and its homologue EupR3 identify a single residue, L285/M261, that controls the production of either 2E,6E,9E- or 2Z,6E,9E-humulene. A possible mechanism for the observed stereoselectivity was investigated using different isoprenoid precursors and results demonstrate that M261 has gatekeeping control over product formation.

AB - The non-canonical terpene cyclase AsR6 is responsible for the formation of 2E,6E,9E-humulene during the biosynthesis of the tropolone sesquiterpenoid (TS) xenovulene A. The structures of unliganded AsR6 and of AsR6 in complex with an in crystallo cyclized reaction product and thiolodiphosphate reveal a new farnesyl diphosphate binding motif that comprises a unique binuclear Mg 2+-cluster and an essential K289 residue that is conserved in all humulene synthases involved in TS formation. Structure-based site-directed mutagenesis of AsR6 and its homologue EupR3 identify a single residue, L285/M261, that controls the production of either 2E,6E,9E- or 2Z,6E,9E-humulene. A possible mechanism for the observed stereoselectivity was investigated using different isoprenoid precursors and results demonstrate that M261 has gatekeeping control over product formation.

KW - biosynthesis

KW - enzyme engineering

KW - humulene

KW - meroterpenoid

KW - tropolone sesquiterpenoid

UR - http://www.scopus.com/inward/record.url?scp=85112167939&partnerID=8YFLogxK

U2 - 10.1002/anie.202106718

DO - 10.1002/anie.202106718

M3 - Article

VL - 60

SP - 20308

EP - 20312

JO - Angewandte Chemie - International Edition

JF - Angewandte Chemie - International Edition

SN - 1433-7851

IS - 37

ER -