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Structure function analysis of benzalacetone synthase from Rheum palmatum
Tsuyoshi Abe1, Hiroyuki Morita, Hisashi Noma
1School of Pharmaceutical Sciences, The COE21 Program, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.
Bioorganic & Medicinal Chemistry Letters
|March 27, 2007
Summary
Benzalacetone synthase (BAS), a plant enzyme, forms diketides via condensation. Mutational analysis revealed specific amino acid substitutions impact BAS activity, suggesting alternative substrate binding pockets in BAS compared to chalcone synthase.
Area of Science:
- Biochemistry
- Plant Molecular Biology
- Enzymology
Background:
- Benzalacetone synthase (BAS) is a plant-specific enzyme belonging to the chalcone synthase (CHS) superfamily of type III polyketide synthases (PKS).
- BAS catalyzes the decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to form diketides.
- The unique diketide-forming activity of Rheum palmatum BAS is linked to a substitution of the conserved active-site Phe215 with Leu.
Purpose of the Study:
- To investigate the structure-function relationship of Rheum palmatum BAS.
- To elucidate the role of specific active-site residues in BAS enzymatic activity and substrate binding.
Main Methods:
- Site-directed mutagenesis was employed to generate four BAS mutants: C197T, C197G, G256L, and S338V.
- Enzymatic assays were performed to assess the activity of the wild-type and mutant BAS enzymes.
- Homology modeling was utilized to predict the structural basis of BAS function.
Main Results:
- All generated mutants retained the characteristic product pattern of BAS.
- The S338V mutant exhibited a 2-fold increase in activity, while the G256L mutant showed a 50% reduction in activity.
- The C197 mutants displayed activity comparable to wild-type BAS, indicating Cys197 is not essential for catalysis.
- Homology modeling suggested BAS may use an alternative pocket for coumaroyl moiety binding, differing from CHS.
Conclusions:
- Specific amino acid residues, particularly at positions 338 and 256, significantly modulate BAS enzymatic activity.
- The conserved Cys residue at position 197 is not critical for the catalytic function of BAS.
- BAS likely employs a distinct substrate-binding mechanism compared to CHS, involving an alternative pocket for the coumaroyl substrate.
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