Properties of a "Split-and-Stuttering" Module of an Assembly Line Polyketide Synthase

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Assembly line polyketide synthases (PKSs) typically follow a "one-module-one-elongation-cycle" rule.
• Some PKS modules exhibit iterative elongation, or "stuttering," but the underlying mechanisms are not fully understood.
• Module iteration offers insights into PKS catalytic flexibility and product diversity.

Purpose of the Study:

• To investigate the intrinsic catalytic properties of a stuttering module from the *trans*-AT PKS subfamily.
• To characterize Module 5 of the NOCAP (Nocardiosis Associated Polyketide) synthase, which is naturally split across two polypeptides.
• To understand the mechanistic principles of module iteration in a unique, split PKS system.

Main Methods:

• In vitro enzymatic assays to determine catalytic activity.
• In vivo studies in *E. coli* to assess module function in a cellular context.
• Analysis of polyketide products to determine elongation cycles and stereochemistry.

Main Results:

• The split stuttering module of NOCAP synthase catalyzed up to five elongation cycles.
• The dehydratase domain of the module ceased function after three cycles.
• Truncated products unexpectedly exhibited a *cis* configuration in the central olefinic group.

Conclusions:

• The study provides the first detailed analysis of a split, stuttering PKS module.
• Findings reveal unexpected catalytic plasticity and stereochemical outcomes in PKS module iteration.
• This work lays the foundation for further exploration of structurally and functionally unusual PKS modules.