STRUCTURE AND FUNCTION OF ITERATIVE POLYKETIDE SYNTHASES (IPKSS) AND HYBRID IPKS–NONRIBOSOMAL PEPTIDE SYNTHETASE (IPKS-NRPS) SYSTEMS INVOLVED IN ANTIBIOTIC BIOSYNTHESIS

Polyketides are structurally diverse natural products with important pharmacological activities, including antibiotic, antifungal, and anticancer properties. These molecules are biosynthesized by polyketide synthases (PKSs), which can be classified into modular or iterative systems. In modular PKSs, each module performs a single cycle of chain elongation. In contrast, iterative PKSs (iPKSs) use the same set of catalytic domains multiple times to construct the polyketide backbone. The final structure is then modified by tailoring enzymes, leading to the bioactive compound.

Some polyketide biosynthetic pathways involve hybrid systems, where iPKS domains are combined with non-ribosomal peptide synthetase (NRPS) modules. An example is the biosynthesis of thiolactomycin (TLM), a thiotetronate antibiotic that targets the type II fatty acid synthase (FAS II) system in bacteria. TLM is produced by a hybrid enzyme called TlmB, which harbors both iPKS and NRPS functionalities within a single polypeptide chain. TlmB catalyzes the formation of the TLM core structure, but the molecular mechanisms underlying its activity remain poorly understood.

TlmB is a large, multifunctional protein of approximately 315 kDa. Due to its size and complexity, structural studies of the full-length enzyme have proven difficult. Previous attempts using cryo-electron microscopy were limited by issues related to protein aggregation. To address these limitations, our group adopted a domain-focused approach, dividing TlmB into the N-terminal iPKS portion, the C-terminal NRPS portion, and additional individual domains. This approach enables isolated expression and purification of smaller individual regions, which are more amenable to structural and biochemical studies using techniques such as NMR spectroscopy, and other biophysical techniques.

We are currently focusing on the acyl carrier protein (ACP), cyclization (Cy) domain, and the NRPS portion to investigate ACP–domain interactions. For this, we are producing isotopically labeled holo-ACP for solution NMR spectroscopy. Our plan includes chemical shift perturbation (CSP) experiments to monitor residue-level changes upon binding to the Cy domain and NRPS portion. This will provide insight into how the ACP delivers the polyketide chain to the Cy domain during TLM biosynthesis. Similar approaches have been applied successfully to study protein–protein interactions in NRPS system, for example by Frueh, Arthanari et al. (Nature 2008;454(7206):903-6). Understanding the interaction dynamics between the ACP and its partner domains will shed light on the association and conformational rearrangements that occur during polyketide transfer and ring formation. These findings will be key to elucidating how hybrid iPKS–NRPS enzymes orchestrate complex chemical transformations and may contribute to the rational engineering of biosynthetic systems for novel antibiotic production.