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. 2011 Sep 23;18(9):1075-81.
doi: 10.1016/j.chembiol.2011.07.016.

Acyl-CoA subunit selectivity in the pikromycin polyketide synthase PikAIV: steady-state kinetics and active-site occupancy analysis by FTICR-MS

Shilah A Bonnett  1 , Christopher M Rath, Abdur-Rafay Shareef, Joanna R Joels, Joseph A Chemler, Kristina Håkansson, Kevin Reynolds, David H Sherman
Affiliations

Acyl-CoA subunit selectivity in the pikromycin polyketide synthase PikAIV: steady-state kinetics and active-site occupancy analysis by FTICR-MS

Shilah A Bonnett et al. Chem Biol. .

Abstract

Polyketide natural products generated by type I modular polyketide synthases (PKSs) are vital components in our drug repertoire. To reprogram these biosynthetic assembly lines, we must first understand the steps that occur within the modular "black boxes." Herein, key steps of acyl-CoA extender unit selection are explored by in vitro biochemical analysis of the PikAIV PKS model system. Two complementary approaches are employed: a fluorescent-probe assay for steady-state kinetic analysis, and Fourier Transform Ion Cyclotron Resonance-mass spectrometry (FTICR-MS) to monitor active-site occupancy. Findings from five enzyme variants and four model substrates have enabled a model to be proposed involving catalysis based upon acyl-CoA substrate loading followed by differential rates of hydrolysis. These efforts suggest a strategy for future pathway engineering efforts using unnatural extender units with slow rates of hydrolytic off-loading from the acyltransferase domain.

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Figures

Figure 1
Figure 1. Catalytic cycle for PikAIV
Acyl-CoA extender units (1 native, 2–4 unnatural) are loaded onto the AT active-site serine as esters (step I) and undergo transfer to the ACP phosphopantetheine producing thioesters (grey portion of CoAS, step II). The hexaketide chain elongation intermediate (5) condenses with the MM-CoA extender unit (steps III–IV) to form the heptaketide (6) on the ACP prior to TE cyclization (Akey et al., 2006) to form narbonolide (7, step V) processed to pikromycin (8, step VI, Li et al., 2009) Reversible steps are noted by a backwards arrow. Off-pathway reactions (ex. hydrolysis from the active-site residues) are not illustrated.
Figure 2
Figure 2. A model for acyl-CoA extender unit processing in the terminal PikAIV PKS module
Arrows represent proposed flux through the system based on Thiolglo steady-state kinetic analysis. Presence/absence of intermediates, as indicated by black/grey coloration, was determined from FTICR-MS analysis of active-site occupancy.
See this image and copyright information in PMC

References

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