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Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase
Paul F Long1, Christopher J Wilkinson, Christian P Bisang
1Cambridge Centre for Molecular Recognition and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
Molecular Microbiology
|March 29, 2002
Summary
Chain initiation in polyketide synthases involves acyl transfer and decarboxylation. Key arginine residues in acyltransferase domains are crucial for this process, enabling specific starter unit incorporation.
Area of Science:
- Biochemistry
- Molecular Biology
- Synthetic Biology
Background:
- Modular polyketide synthases (PKS) assemble complex molecules.
- Chain initiation often involves acyl transfer from a dicarboxylic acid CoA ester, followed by decarboxylation by a ketosynthase-like decarboxylase (KSQ) domain.
- Acyltransferase (AT) domains within PKS loading modules are critical for selecting and transferring starter units.
Purpose of the Study:
- To investigate the role of a specific arginine residue in the AT domains of KSQ-containing loading modules.
- To determine the impact of altering loading module AT domains on polyketide product profiles.
- To confirm the mechanism of direct acylation of the ketosynthase domain in chain initiation.
Main Methods:
- Site-directed mutagenesis of the arginine residue in the oleandomycin (ole) loading AT domain.
- Heterologous expression and functional analysis of engineered PKS constructs.
- Analysis of polyketide products derived from wild-type and mutant PKS systems.
- Construction and characterization of a mutant triketide synthase (DEBS1-TE) lacking the phosphopantetheine attachment site.
Main Results:
- Replacement of the key arginine residue in the ole loading AT domain abolished its activity.
- Swapping loading modules between different PKS (ole, tylosin, erythromycin) altered the starter unit incorporation, yielding predominantly acetate- or propionate-derived products.
- An engineered rapamycin PKS AT domain (rap AT2) successfully directed the synthesis of C13-methylerythromycins when placed in the ole loading module.
- A mutant DEBS1-TE lacking the 4'-phosphopantetheine attachment site produced triketide lactone products, confirming direct acylation of the ketosynthase.
Conclusions:
- The active site arginine residue in KSQ-containing AT domains is essential for catalysis.
- The loading module AT domain dictates the type of starter unit incorporated into polyketides.
- Direct acylation of the ketosynthase domain of extension module 1 plays a confirmed role in chain initiation.