10.1002/chem.202004444
Chemistry - A European Journal
FULL PAPER
that expression of early genes from cytochalasan pathways in A.
oryzae always leads to alcohol shunt intermediates.
Corroborating observations from Lebrun and coworkers show
that ORFZ (hydrolase) and ORF3 (Diels Alderase) are the most
highly expressed genes from the ACE1 BGC (up to 7-fold higher
Acknowledgements
The authors would like to thank Dr. Jennifer Senkler and Prof. Dr.
Hans-Peter Braun from the Institute of Plant Genetics, Leibniz
University of Hanover for protein sequence analysis by ESI Q-
TOF. Inken Hertrampf (LUH) and Franck Siacku (LUH) are
thanked for technical assistance. DFG is thanked for the
provision of NMR and LCMS equipment (INST 187/621-1, INST
187/686-1). VH was funded by DFG (CO 1328/2-1) and HZ was
funded by the China Scholarship Council (CSC 201506200065).
than the ACE1 PKS-NRPS itself), suggesting
a
high
concentration of these catalysts is required to avoid the
reductive shunt pathways.[10]
Second, the model studies show that rapid tautomerisation
of 24a to 24b creates a substrate which is incompetent for
further Diels Alder reaction because the alkene is no longer
present at C-3,C-4. Tautomer 24b is itself reactive and
undergoes other reactions leading it away from the cytochalasin
pathway. Interestingly the observed hemi-aminal functionality of
25ab mirrors the same functionality found in in fusarin C[18] and
talaroconvolutin B,[35] for example.
Keywords: hydrolase • pyrrolone • Knoevenagle • cytochalasan
• biosynthesis
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These conclusions show that the previously widely held
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It is intriguing that the PyiE enzyme family preserve the
nucleophilic active site serine at a ‘nucleophilic elbow’ position
found in DHPON hydrolase and AntI, but lack the acid/base
residues required for retro-Claisen reaction. However the
acid/base residues shown by Bode and coworkers to be
involved in cyclisation in AntI are conserved in PyiE and ORFZ.
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and its homologues could involve addition of the serine
nucleophile of PyiE/ORFZ to the b-ketone of 20 during reaction
(Scheme 6C) to give an enzyme-bound tetrahedral intermediate,
without retro Claisen cleavage because of the lack of the
catalytic triad H and D residues. The formation of a tetrahedral
center at the b-position would prevent incorrect tautomerisation,
providing the correct (enzyme-bound) substrate for the
subsequent Diels Alder reaction. This in-turn suggests that the
hydrolase (e.g. PyiE/ORFZ) and Diels-Alderase (e.g.
PyiF/ORF3) catalysts may work closely together or as a hetero-
dimer, or conceivably with the PKS-NRPS itself. However,
further detailed in vitro experiments will be required to test this
idea, and more reliable methods for the production of soluble
and active PyiE/ORFZ and PyiF/ORF3 proteins will be required
to probe this hypothesis further. However synthetic and analytic
progress made here sets the scene for future advances in this
area.
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See supplementary information for full experimental details, structural
characterization data, NMR spectra, chromatograms etc.
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