Combinatorial biosynthesis of polyketides through engineering the respective biosynthetic pathways represents a promising approach to natural products discovery. However, a lack of information on substrate and stereochemical specificity of polyketide synthases (PKS) currently hinders these efforts. The dehydratase (DH) domains of PKS, which generate an α,β-unsaturated bond through dehydration of a b-alcohol, have extremely high stereospecficity towards substrates and can prematurely terminate the nascent polyketide when presented with unnatural substrates. We have selected the DH domain from pikromycin module 2 as a model system, which is responsible for dehydration of a triketide chain intermediate. We will report the cloning, overexpression, and biochemical characterization of PikDH2 and point mutants of active site residues important for catalysis and substrate recognition. For this study, mimics of the natural chain intermediates were synthesized and enzyme activity was evaluated using an LC-MS/MS assay to monitor product formation (Figure 1). By using our probes, we demonstrated that PikDH2 processes only D-alcohols, and not L-alcohols. In addition to the β-position, the enzyme showed extremely high stereospecificity on the γ- and δ-stereocenters, with only the native configuration leading to product formation. Through the pH rate profile, we determined a p<i>K</i><sub>a</sub> of 6.9 for the catalytic residue, consistent with the expectation that a conserved histidine is the catalytic base. Initial mutagenesis studies and kinetic analysis have indicated the importance of two phenylalanine residues (F172 and F176), which may shape the hydrophobic region of the active site. This study reveals that distal stereogenic centers of the PikDH2 substrate may either participate in binding the active site or assist in adopting a catalytically competent conformation. We also identify several hydrophobic residues that are critical for high enzymatic activity. Through a combination of point mutations and substrate permutations, our studies aim to uncover the native docking pose and key residues important in native PKS catalytic events, as well as aid in future combinatorial biosynthetic efforts.

Functional characterization of a polyketide synthase (PKS) dehydratase domain using chemical probes

249th National Meeting (2015)

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