An Oxetane-Based Polyketide Surrogate To Probe Substrate Binding in a Polyketide Synthase

Abstract

Polyketides are a large class of bioactive natural products with a wide range of structures and functions. Polyketides are biosynthesized by large, multidomain enzyme complexes termed polyketide synthases (PKSs). One of the primary challenges when studying PKSs is the high reactivity of their poly-β-ketone substrates. This has hampered structural and mechanistic characterization of PKS-polyketide complexes, and, as a result, little is known about how PKSs position the unstable substrates for proper catalysis while displaying high levels of regio- and stereospecificity. As a first step toward a general plan to use oxetanes as carbonyl isosteres to broadly interrogate PKS chemistry, we describe the development and application of an oxetane-based PKS substrate mimic. This enabled the first structural determination of the acyl-enzyme intermediate of a ketosynthase (KS) in complex with an inert extender unit mimic. The crystal structure, in combination with molecular dynamics simulations, led to a proposed mechanism for the unique activity of DpsC, the priming ketosynthase for daunorubicin biosynthesis. The successful application of an oxetane-based polyketide mimic suggests that this novel class of probes could have wide-ranging applications to the greater biosynthetic community interested in the mechanistic enzymology of iterative PKSs.

Computational structural enzymology methodologies for the study and engineering of fatty acid synthases, polyketide synthases and nonribosomal peptide synthetases

“…The free energy calculations show that the native malonatebased substrate and the oxetane-based substrate had similar ΔG binding with no statistically significant differences in their calculated binding affinities. PCA was used to evaluate sampling of the receptor with either substrate present, showing sufficient overlap (Ellis et al, 2018). The above result is promising and the syntheses of higher-order poly-β-ketone mimics (tetra-to dodeca-ketides) are currently underway, which will greatly aid in the visualization of PKSs in action (Tsai, 2018).…”

“…This method involves the calculation of (1) the solvation free energy contribution by solving the linearized Poisson Boltzmann or Generalized Born equation, (2) the gas phase energy contribution by performing a molecular mechanics calculation, and (3) the entropy contribution by performing normal mode analysis. In our group, Ellis et al performed MM-PBSA to calculate the binding free energy between the enzyme DpsC and its native substrate or the recently developed oxetane-based substrate mimics in order to assess the validity of the mimics (Section 2.2.3) (Ellis et al, 2018). MM-PBSA has also been used to assess the contributions of interface residues in FAS (Section 2.1.3).…”

“…We recently designed and synthesized oxetane-polyketide mimics using oxetanes as carbonyl isosteres (Fig. 10B-C) (Ellis et al, 2018;Tsai, 2018). Oxetanes have been shown to mimic both the geometry and dipole properties of carbonyl groups, while sterically preventing intramolecular aldol cyclization at the same time (Bull, Croft, Davis, Doran, & Morgan, 2016;Ellis et al, 2018;Luger & Buschmann, 1984).…”

A Light‐Activated Acyl Carrier Protein “Trap” for Intermediate Capture in Type II Iterative Polyketide Biocatalysis

“…More recent biophysical studies (e.g., X‐ray crystallography and NMR) of type II PKS proteins have provided more in‐depth knowledge on protein/ substrate recognition and productive conformations. Some of these investigations have relied on the enzymatic loading of synthetically prepared intermediate mimics of ACPs, as the natural ACP‐bound intermediates are intrinsically unstable and highly reactive . The general inability of directly monitoring iterative intermediate formation and processing in real time constitutes a significant hurdle in gathering new knowledge of enzyme kinetics and protein‐substrate interactions required to devise novel synthetic biology .…”

“…Someo f these investigationsh ave relied on the enzymatic loading of synthetically prepared intermediate mimics of ACPs, as the natural ACP-bound intermediates are intrinsically unstablea nd highly reactive. [15,16] The general inability of directly monitoring iterative intermediate formation and processing in real time constitutes as ignificant hurdle in gathering new knowledgeo f enzymek inetics and protein-substrate interactions required to devise novel syntheticb iology. [17] In our labs we have established ac hemical 'chain termination" methodology aimed at the captureo ft ransientp olyketide biosynthetic intermediates in vitro [18] and in vivo.…”