Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae

被引:13
作者
Lukacin, R
Schreiner, S
Silber, K
Matern, U
机构
[1] Univ Marburg, Inst Pharmazeut Biol, D-35037 Marburg, Germany
[2] Univ Marburg, Inst Pharmazeut Chem, D-35032 Marburg, Germany
关键词
Ruta graueolens L; Dictaninus albus; rutaceaee; acridone synthase; chalcone synthase; site-directed mutagenesis;
D O I
10.1016/j.phytochem.2004.11.023
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. ACSs which have been cloned exclusively from Ruta graveolens share about 75-85% polypeptide sequence homology with CHSs from other plant families, while 90% similarity was observed with CHSs from Rutaceae, i.e., R. graveolens, Citrus sinensis and Dictamnus albus. CHSs cloned from many plants do not accept N-methylanthraniloyl-CoA as a starter substrate, whereas ACSs were shown to possess some side activity with 4-coumaroyl-CoA. The transformation of an ACS to a functional CHS with 10% residual ACS activity was accomplished previously by substitution of three amino acids through the corresponding residues from Ruta-CHS1 (Ser132Thr, Ala133Ser and Val265Phe). Therefore, the reverse triple mutation of Ruta-CHS1 (mutant R2) was generated, which 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 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 wildtype 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 R. 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. (C) 2004 Elsevier Ltd. All rights reserved.
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收藏
页码:277 / 284
页数:8
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