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Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes
Published on: January 16, 2016
Structure-based engineering of benzalacetone synthase
Yoshihiko Shimokawa1, Hiroyuki Morita, Ikuro Abe
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
Bioorganic & Medicinal Chemistry Letters
|July 30, 2010
Summary
Mutating benzalacetone synthase (BAS) active-site residue Leu132 to Thr restored chalcone formation. Other mutations, like L132A, produced novel compounds, revealing insights into type III polyketide synthase (PKS) mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide synthases (PKSs) with homologous structures.
- BAS produces diketide benzalacetone, while CHS produces tetraketide chalcone via iterative malonyl-CoA condensations.
- Active-site residue substitutions in BAS, specifically Leu132, are hypothesized to cause steric contraction, favoring diketide production.
Purpose of the Study:
- To investigate the mechanistic consequences of site-directed mutations at the Leu132 residue in Rheum palmatum BAS.
- To determine how altering the active-site cavity affects product chain length and aromatic ring formation.
Main Methods:
- Site-directed mutagenesis of R. palmatum BAS to create mutants L132G, L132A, L132S, L132C, L132T, L132F, L132Y, L132W, and L132P.
- Enzymatic assays to analyze the products formed by BAS mutants.
- Homology modeling to predict structural changes in the active-site cavity.
Main Results:
- The L132T mutation restored chalcone-forming activity in BAS.
- Mutations L132A, L132S, and L132C resulted in the production of 4-coumaroyltriacetic acid lactone (CTAL), indicating expanded product chain length without aromatic ring formation.
- Homology modeling suggested restoration of the 'coumaroyl binding pocket' for CTAL-producing mutants.
Conclusions:
- The Thr132 residue in CHS is crucial for tetraketide and aromatic ring formation, while Leu132 in BAS favors diketide production due to steric effects.
- Specific amino acid substitutions at position 132 can modulate the catalytic activity and product specificity of type III PKS enzymes.
- These findings enhance the understanding of the structure-function relationships and catalytic mechanisms of type III PKSs.
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