Browsing by Subject "Polyketide synthase"
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Item Enzymatic features and biocatalytic applications of modular polyketide synthase domains(2015-12) Bailey, Constance Beryl; Keatinge-Clay, Adrian Tristan; Anslyn, Eric V; Liu, Hung-Wen; Hoffman, David; Garneau-Tsodikova, SylviePolyketides are a class of secondary metabolites that are notable for their chemical diversity and therapeutic relevance. They are biosynthesized by polyketide synthases (PKSs) megasynthase enzymes in an assembly-line fashion. Though the molecular architectures of polyketides are complex, their biological precursors are chemically simple. Thus, understanding this powerful biosynthetic machinery is of interest for synthetic biology and biocatalytic applications. This dissertation presents three projects that decipher underlying mechanistic features and explore biocatalytic applications of PKSs. In modular PKSs, one module corresponds to one round of keto-elongation followed by modification through the action of β-carbon processing domains. The first project employs a system wherein a single module is used in vitro to generate small, chiral PKS products (triketide lactones). Although triketide lactones are a common output for PKS enzymology assays, usually they are only observed in trace quantities. In this study, we performed a number of strategies to scale up the production of triketide lactones to facilitate their use as chiral building blocks for chemical synthesis. In this process, we also gained new insights regarding the interacting kinetics and selectivities of the domains in an in vitro environment. ix The second project focused on the ketoreductase (KR) domain, which sets the majority of the stereogenic centers within a polyketide, and thus has obvious potential for biocatalytic applications. This project employs a structure-activity relationship (SAR)- type approach to dissecting stereocontrol. The SAR results, in concert with crystallographic data inspired two rational mutations that were sufficient to reverse the stereoselectivity of a representative KR. Thus, we were able to employ a rational approach to engineering stereocontrol. The final project also focuses on the KR domain, however from a subclass of PKSs termed trans-acyltranferase (AT) PKSs. In contrast to the canonical cis-AT PKSs, the trans-AT PKSs have more varied modular organizations and architectures. One of these peculiar organizations one termed a “split” bimodule, wherein domains within a module are present on different polypeptides. Structural characterization of a KR from a split bimodule revealed features that may correspond to interpeptide interactions that afford communication between the two polypeptides of the split bimodule. Additionally, bioinformatic analysis of KRs from split bimodules reveals a number of diagnostic sequence motifs.Item In vitro polyketide biocatalysis : triketide building-blocks and enzymology(2013-05) Harper, Andrew David; Keatinge-Clay, Adrian TristanPolyketide products are useful compounds to research and industry but can be difficult to access due to their richness in stereogenic centers. Type I polyketide synthases offer unique engineering opportunities to access natural stereocontrol and resultant complex compounds. The development of a controlled in vitro platform based around type I polyketide synthases is described. It has been used to produce a small library of polyketide fragments on an unprecedented and synthetically-relevant scale and explore polyketide synthase enzymology.Item Investigations into the biocatalytic potential of modular polyketide synthase ketoreductases(2013-08) Piasecki, Shawn Kristen; Keatinge-Clay, Adrian TristanThe 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.Item Towards preparative in vitro enzymatic synthesis of new polyketide metabolites(2013-08) Hughes, Amanda Jane; Keatinge-Clay, Adrian TristanModular polyketide synthases (PKSs) are the largest enzymes known to man and are responsible for synthesizing some of the most important human medicines. Their ability to construct stereochemically-rich carbon chains containing diverse substituents has inspired the biosynthetic community to engineer these factories for the in vitro synthesis of a small library of polyketide compounds. New complex polyketides are discovered every year, yet the lack of compound prohibits characterization and testing of these new compounds for medicinal properties. Smaller polyketide compounds generated in vitro could be organically manipulated to generate larger, more complex polyketide natural products and natural product analogs. Chemoenzymatic approaches like this would be extremely beneficial to the scientific community; however, there are still obstacles that must be overcome before the use of PKS for the preparative synthesis of an in vitro generated polyketide library would prove fruitful: purchasing substrates such as methylmalonyl-CoA is cost-prohibitive, PKSs are often difficult to express and purify, and the products generated are typically nonchromophoric. The use of a malonyl-CoA ligase from Streptomyces coelicolor (MatB) was investigated for the enzymatic synthesis of polyketide extender units such as methylmalonyl-CoA (Chapter 2). MatB synthesized a total of 5 CoA-linked extender units in vitro: malonyl-, methylmalonyl-, ethylmalonyl-, hydroxymalonyl- and methoxymalonyl-CoA. Two ternary complex structures of MatB with bound product and leaving group were also solved to sub-2Å resolution. MatB generated extender units were employed in the module-catalyzed synthesis of a triketide pyrone. The selectivity of a PKS module to incorporate a variety of side chains into triketide pyrones was also investigated (Chapter 3). A total of 10 triketide pyrone compounds were synthesized, 5 produced via modular "stuttering" and one possessing a terminal alkyne chemical handle. Lastly, nonchromphoric polyketide products were made visible upon copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) with fluorescent sulforhodamine B azide revealing insights into in vitro reactivites of a PKS module (Chapter 4). The work described in this dissertation has helped advance the scientific community towards procuring an in vitro synthesized polyketide library for future synthetic applications.Item Unveiling the architectures of five bacterial biomolecular machines(2014-08) Fage, Christopher Dane; Keatinge-Clay, Adrian Tristan; Hoffman, David W; Whitman, Christian P; Appling, Dean R; Iverson, Brent L; Hackert, Marvin LNatural products represent an incredibly diverse set of chemical structures and activities. Given this fathomless, ever-evolving diversity, a reasonable approach to designing new molecules entails taking a closer look at the biochemistry that Nature has crafted over billions of years on Earth. In particular, much can be learned by unveiling the architectures of proteins, life’s molecular machines, through methods like X-ray crystallography. Acquiring the blueprints of an enzyme brings us closer to understanding the mechanism by which the enzyme transforms a simple substrate it into a complex product with biological function, and inspires us to engineer such systems to our own ends. With a focus on macromolecular structural characterization, this document elaborates on five Gram-negative bacterial biosynthetic enzymes from two categories: Cell-surface modifiers and polyketide synthases. Among the first category are the glycyl carrier protein AlmF and its ligase AlmE of Vibrio cholerae and the phosphoethanolamine transferase EptC of Campylobacter jejuni. These proteins are responsible for decorating cell-surface molecules (e.g., lipid A) of pathogenic bacteria with small functional groups to promote antibiotic resistance, motility, and host colonization. AlmE and EptC represent potential drug targets and their structures lay the groundwork for the design of therapeutics against food-borne illnesses. Included in the second category are the [4+2]-cyclase SpnF and two ketoreductase-linked dimerization elements, each from the spinosyn biosynthetic pathway in Saccharopolyspora spinosa. The former catalyzes a putative Diels-Alder reaction to form a tricyclic precursor of the insecticide spinosad, while the latter two organize ketoreductase domains within modules of a polyketide synthase. The second category also includes Ralstonia eutropha β-ketoacyl thiolase B, a substrate-permissive enzyme that can make or break carbon-carbon bonds with assistance from Coenzyme A or an analogous thiol. Each of these proteins exhibit intriguing structural features or catalyze reactions that show promise for biochemical engineering.