Structural and functional analysis of the loading acyltransferase from avermectin modular polyketide synthase.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Modular polyketide synthases (PKSs) are crucial for producing diverse polyketide natural products.
• Loading acyltransferase (AT) domains within PKSs dictate the selection of starter units, influencing polyketide structure.
• Engineering AT domains offers a pathway to modify polyketide biosynthesis and generate novel compounds.

Purpose of the Study:

• To elucidate the structural and biochemical properties of the loading AT domain from the avermectin PKS.
• To understand the molecular basis for the relaxed substrate specificity of this loading AT.
• To enable targeted engineering of AT domains for polyketide modification.

Main Methods:

• X-ray crystallography for structural determination of the loading AT.
• Biochemical assays to assess substrate acceptance and specificity.
• Site-directed mutagenesis to alter active site residues.
• Enzymatic hydrolysis assays to confirm stereospecificity.

Main Results:

• The first structural analysis of a modular PKS loading AT revealed the molecular basis for its broad substrate specificity.
• Modeling identified key residues involved in substrate binding and discrimination.
• A mutant AT domain exhibited altered specificity towards synthetic substrate mimics.
• The avermectin loading AT demonstrated stereospecificity for the S-configuration of 2-methylbutyric acid.

Conclusions:

• The structural and biochemical characterization provides a foundation for understanding loading AT function.
• Active site engineering of AT domains can be used to modify polyketide structures.
• This work facilitates the targeted modification of polyketides through PKS engineering.