Chemical and Biochemical Interrogation of Molecular Specificity in Modular Polyketide Synthases.

Polyketide natural products represent a diverse set of chemical entities that are prized by organic and medicinal chemists for their complex molecular architectures and unique pharmacological properties. These natural products are generated as secondary metabolites in a variety of marine and terrestrial microbial sources through the decarboxylative condensation of coenzyme A (CoA) esters of simple malonic acids. These condensation reactions occur on large, modular enzyme complexes called polyketide synthases (PKSs). Two prime examples of these unique multifunctional enzyme systems are the 6-deoxyerythronolide B synthase (DEBS) and pikromycin (Pik) PKS, which are responsible for the biosynthesis of the erythromycin and pikromycin aglycones, respectively. Together, these natural product biosynthetic systems provide excellent platforms to examine the fundamental structural and catalytic elements that govern polyketide assembly, processing and macrocyclization. Due to the modular architecture of bacterial type I PKSs, rational bioengineering of these enzymes has opened up an avenue toward the rapid generation of polyketide chemical diversity through chemoenzymatic synthesis and combinatorial biosynthesis. Realization of this goal, however, requires a detailed understanding of molecular specificity in the catalytic domains of these PKS enzymes. Toward this goal, this dissertation describes the development of synthetic methodologies to access late-stage polyketide chain-elongation intermediates from the DEBS and Pik systems and their subsequent employment in biochemical studies with engineered PKS modules. Specifically, the native pentaketide intermediate for the DEBS system was synthesized and employed for in vitro chemoenzymatic synthesis of macrolactone products in the final engineered monomodules Ery5, Ery5-TE and Ery6 as well as bimodular DEBS3. A comparative analysis was performed with the corresponding Pik module 5 (PikAIII) and module 6 (PikAIV), utilizing native chain Pik chain-elongation intermediates, and dissecting key similarities and differences between these highly related PKSs. The data revealed that individual modules in the DEBS and Pik PKSs possess distinctive molecular selectivity profiles, and suggest that substrate recognition has evolved unique characteristics in each system. Additional work has been put forth to establish a general methodology to access DEBS and Pik penta- and hexaketide analogues that encompass a number of stereochemical, structural and functional group variations.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/77767/1/mortison_1.pd

Tetracyclines Modify Translation by Targeting Key Human rRNA Substructures

International audienceApart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines

Biocatalytic Synthesis of Pikromycin, Methymycin, Neomethymycin, Novamethymycin, and Ketomethymycin

A biocatalytic platform that employs the final two monomodular type I polyketide synthases of the pikromycin pathway in vitro followed by direct appendage of d-desosamine and final C–H oxidation(s) in vivo was developed and applied toward the synthesis of a suite of 12- and 14-membered ring macrolide natural products. This methodology delivered both compound classes in 13 steps (longest linear sequence) from commercially available (<i>R</i>)-Roche ester in >10% overall yields