How cis-Acyltransferase Assembly-Line Ketosynthases Gatekeep for Processed Polyketide Intermediates

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Polyketide synthases (PKS) are crucial for producing diverse polyketide natural products.
• The ketosynthase (KS) domain is the catalytic core of PKS, responsible for carbon-carbon bond formation.
• Recent studies suggest KS domains play a role in selecting and processing polyketide intermediates.

Purpose of the Study:

• To investigate the structural diversity and substrate specificity of ketosynthase (KS) domains.
• To identify sequence fingerprints within KS domains correlated with specific polyketide substrate chemistries.
• To explore the potential for engineering KS domains for novel substrate acceptance.

Main Methods:

• Bioinformatic analysis of 739 KS domains from actinomycete assembly lines.
• Classification of KS domains into families based on substrate α-, β-, and γ-carbon chemistries.
• Molecular modeling of KS domains bound to natural polyketide substrates.
• Site-directed mutagenesis of a conserved glutamine residue in a model triketide synthase KS domain.

Main Results:

• Unique sequence fingerprints were identified in KS substrate tunnel residues, correlating with α- and β-carbon chemistries.
• Additional fingerprints emerged when considering γ-carbon chemistry, revealing finer substrate discrimination.
• Molecular modeling elucidated specific residue substitutions accommodating substrate features like l-α-methyl groups or primer units.
• Mutagenesis demonstrated that altering a conserved glutamine impacts KS function, suggesting substrate tunnel sensitivity.

Conclusions:

• KS domains exhibit remarkable sequence diversity linked to polyketide substrate specificity.
• The KS substrate tunnel is a key determinant of substrate selection and processing.
• Engineering KS domains for unnatural substrates presents a challenge, likely requiring multiple targeted mutations.