Molekulare Untersuchungen zur Spezifität von Polyketidsynthasen aus Dictamnus albus L. und Ruta graveolens L.
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Philipps-Universität Marburg
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Abstract
Chalcone synthases (CHSs) and acridone synthases
(ACSs) belong to the superfamily of type III polyketide
synthases (PKSs) and condense the starter substrate
4-coumaroyl-CoA or N-methylanthraniloyl-CoA with three
malonyl-CoAs to produce flavonoids and acridone alkaloids,
respectively. While acridone alkaloids are confined almost
exclusively to the Rutaceae, flavonoids occur abundantly in all
seed-bearing plants. ACSs and CHSs had been cloned from Ruta
graveolens and shown to be closely related polyketide synthases
which use N-methylanthraniloyl-CoA and 4-coumaroyl-CoA,
respectively, as the starter substrate to produce the acridone
or naringenin chalcone. As proposed for the related 2-pyrone
synthase from Gerbera, the differential substrate specificities
of ACS and CHS might be attributed to the relative volume of
the active site cavities. The primary sequences as well as the
immunological cross reactivities and molecular modeling studies
suggested an almost identical spatial structure for ACS and
CHS. Based on the Ruta ACS2 model the residues Ser132, Ala133
and Val265 were assumed to play a critical role in substrate
specificity. Exchange of a single amino acid (Val265Phe)
reduced the catalytic activity by about 75% but grossly shifted
the specificity towards CHS activity, and site-directed
mutagenesis replacing all three residues by the corresponding
amino acids present in CHS (Ser132Thr, Ala133Ser and Val265Phe)
fully transformed the enzyme to a functional CHS with
comparatively marginal ACS activity. The results suggested that
ACS divergently has evolved from CHS by very few amino acid
exchanges, and it remains to be established why this route of
functional diversity has developed in the Rutaceae only. The
reverse triple mutation of Ruta-CHS1 (mutant R2) affected only
insignificantly the CHS activity and did not confer ACS
activity. However, competitive inhibition of CHS activity by
N-methylanthraniloyl-CoA was observed for the mutant and in
contrast to wild-type CHSs. Homology modeling of ACS2 with
docking of 1,3-dihydroxy-N-methylacridone suggested that the
starter substrates for CHS or ACS reaction are placed in
different topographies in the active site pocket. Additional
site specific substitutions (Asp205Pro/Thr206Asp/His207Ala or
Arg60Thr and Val100Ala/ Gly218Ala, respectively) diminished the
CHS activity to 75%-50% of the wild-type CHS1 without promoting
ACS activity. The results suggest that conformational changes
in the periphery beyond the active site cavity volumes
determine the product formation by ACSs vs. CHSs in Ruta
graveolens. It is likely that ACS has evolved from CHS, but the
sole enlargement of the active site pocket as in CHS1 mutant R2
is insufficient to explain this process.
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Metadata
Contributors
Supervisor:
Dates
Created: 2004Issued: 2004-05-19Updated: 2011-08-10
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
ACSRuta graveolens , Dictamnus albusACSpolyketideCHSCHSacridone synthase , chalcone synthase
DFG-subjects
RautengewächsePolyketide , SekundärstoffwechselFlavonoideFlavonoidstoffwechsel
DDC-Numbers
610
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Schreiner, Stephan: Molekulare Untersuchungen zur Spezifität von Polyketidsynthasen aus Dictamnus albus L. und Ruta graveolens L.. : Philipps-Universität Marburg 2004-05-19. DOI: https://doi.org/10.17192/z2004.0127.
License
This item has been published with the following license: In Copyright