Molekulare Untersuchungen zur Spezifität von Polyketidsynthasen aus Dictamnus albus L. und Ruta graveolens L.

Ziel dieser Arbeit war es, einen Beitrag zur Aufklärung der molekularen Funktionsweise von Polyketidsynthasen (PKS) zu leisten. Hierzu wurden Mutagenesestudien sowie biochemische Untersuchungen an Chalkonsynthase (CHS) und Acridonsynthase (ACS) aus Ruta graveolens durchgeführt. N...

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Bibliographic Details
Main Author: Schreiner, Stephan
Contributors: Prof. Dr. U. Matern (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2004
Online Access:PDF Full Text
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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.