Employing Modular Polyketide Synthase Ketoreductases as Biocatalysts in the Preparative Chemoenzymatic Syntheses of Diketide Chiral Building Blocks

SummaryChiral building blocks are valuable intermediates in the syntheses of natural products and pharmaceuticals. A scalable chemoenzymatic route to chiral diketides has been developed that includes the general synthesis of α-substituted, β-ketoacyl N-acetylcysteamine thioesters followed by a biocatalytic cycle in which a glucose-fueled NADPH-regeneration system drives reductions catalyzed by isolated modular polyketide synthase (PKS) ketoreductases (KRs). To identify KRs that operate as active, stereospecific biocatalysts, 11 isolated KRs were incubated with 5 diketides and their products were analyzed by chiral chromatography. KRs that naturally reduce small polyketide intermediates were the most active and stereospecific toward the panel of diketides. Several biocatalytic reactions were scaled up to yield more than 100 mg of product. These syntheses demonstrate the ability of PKS enzymes to economically and greenly generate diverse chiral building blocks on a preparative scale

Investigations into the biocatalytic potential of modular polyketide synthase ketoreductases

textThe production of new drugs as potential pharmaceutical targets is arguably one of the most important avenues of medicine, as existing diseases not only require treatment, but it is also certain that new diseases will appear in the future which will need treatment. Indeed, existing medicines such as antibiotics and immunosuppressants maintain their current activities in their respective realms, yet the molecular and stereochemical complexity of these compounds cause a burden on organic synthetic chemists that may prohibit the high yields required to manufacture a drug. The enzyme systems that naturally manufacture these compounds are incredibly efficient in doing so, and also do not use environmentally harmful solvents, chiral auxiliaries, or metals that are utilized in the current syntheses of these compounds; therefore utilizing these enzymes' machinery for the biocatalysis of new medicinally-relevant compounds, as researchers have in the past, is undeniably a rewarding endeavor. In order to harness these systems' biocatalytic potential, we must understand the processes which they operate. This work focuses on ketoreductase domains, since they are responsible for setting most of the stereocenters found within these complex secondary metabolites. We have supplied a library of substrates to multiple ketoreductases to test their limits of stereospecificity and found that, for the most part, they maintain their natural product stereospecificity seen in nature. We were even able to convert a previously nonstereospecific ketoreductase to a stereospecific catalyst. We have also developed a new technique to follow ketoreductase catalysis in real-time, which can also differentiate between which diastereomeric product is being produced. Finally, we have elucidated the structure of a ketoreductase that reduces non-canonically at the [alpha]- and [beta]- position, and functionally characterized its activities on shortened substrate analogs. With the knowledge gained from this dissertation we hope that the use of ketoreductases as biocatalysts in the biosynthesis of new natural product-based medicines is a much nearer reality than before.Cellular and Molecular Biolog

Theory, Experiment and Computer Simulation of the Electrostatic Potential at Crystal/Electrolyte Interfaces

In this feature article we discuss recent advances and challenges in measuring, analyzing and interpreting the electrostatic potential development at crystal/electrolyte interfaces. We highlight progress toward fundamental understanding of historically difficult aspects, including point of zero potential estimation for single faces of single crystals, the non-equilibrium pH titration hysteresis loop, and the origin of nonlinearities in the titration response. It has been already reported that the electrostatic potential is strongly affected by many second order type phenomena such as: surface heterogeneity, (sub)surface transformations, charge transfer reactions, and additional potential jumps at crystal face edges and/or Schottky barriers. Single-crystal electrode potentials seem particularly sensitive to these phenomena, which makes interpretation of experimental observations complicated. We hope that recent theory developments in our research group including an analytical model of titration hysteresis, a perturbative surface potential expansion, and a new surface complexation model that incorporates charge transfer processes will help experimental data analysis, and provide unique insights into the electrostatic response of nonpolarizable single-crystal electrodes

Structural Studies of an A2-Type Modular Polyketide Synthase Ketoreductase Reveal Features Controlling α‑Substituent Stereochemistry

Modular polyketide synthase ketoreductases often set two stereocenters when reducing intermediates in the biosynthesis of a complex polyketide. Here we report the 2.55-Å resolution structure of an A2-type ketoreductase from the 11th module of the amphotericin polyketide synthase that sets a combination of l-α-methyl and l-β-hydroxyl stereochemistries and represents the final catalytically competent ketoreductase type to be structurally elucidated. Through structure-guided mutagenesis a double mutant of an A1-type ketoreductase was generated that functions as an A2-type ketoreductase on a diketide substrate analogue, setting an α-alkyl substituent in an l-orientation rather than in the d-orientation set by the unmutated ketoreductase. When the activity of the double mutant was examined in the context of an engineered triketide lactone synthase, the anticipated triketide lactone was not produced even though the ketoreductase-containing module still reduced the diketide substrate analogue as expected. These findings suggest that re-engineered ketoreductases may be catalytically outcompeted within engineered polyketide synthase assembly lines