Biochemical and structural studies on proteins involved in the production of natural products and degradation of polysaccharides

Chekan, Jonathan Rodi

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Description

Title

Biochemical and structural studies on proteins involved in the production of natural products and degradation of polysaccharides

Author(s)

Chekan, Jonathan Rodi

Issue Date

2016-10-31

Director of Research (if dissertation) or Advisor (if thesis)

Nair, Satish K.

Doctoral Committee Chair(s)

Nair, Satish K.

Committee Member(s)

• Cronan, John E.
• Jin, Hong
• Metcalf, William W.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Natural products
• Crystallography
• Biochemistry
• Ribosomally synthesized and post-translationally modified peptides (RiPPs)

Abstract

One of the major limitations of traditional organic chemical synthesis is the challenge of modifying a starting material with both stereoselectivity and regioselectivity to produce a single product. Nature has solved this problem by evolving enzymes that specifically bind ligands and orientate them for selective modification. Furthermore, enzymes are generally active under mild conditions, forgoing the harsh chemicals and byproducts often necessary in chemical synthesis. Therefore, there has been a growing interest to utilize enzymes as an alternative method for the production of a variety of compounds. Organic chemistry is limited is in the production of natural products. Natural products are broadly defined as compounds produced by living organisms that have bioactivity. With activities including antibacterial, antimycotic, antimalarial, cytotoxic, and immunosuppressive, natural products represent a plentiful source of medically relevant compounds. However, the synthesis of these molecules is often difficult due to their large size and complexity. For example, the common anticancer compound taxol is produced by isolating the immediate precursor from its natural source, the Pacific yew tree, because the total organic synthesis is prohibitively expensive. To effectively utilize enzymes in the production of complex natural products or biofuels, it is important to understand the molecular basis for their catalysis. This information can allow for the design of new enzymes that create novel compounds or the prediction of existing natural products by examining sequenced genomes. In this dissertation, I will present my efforts to understand both the ligand binding and catalytic mechanisms of proteins involved in the biosynthesis of RiPP natural products and polysaccharide degradation. RiPP (ribosomally synthesized and posttranslationally modified peptide) natural products represent a growing class of compounds. They are biosynthesized from a linear precursor peptide that was translated by the ribosome. A series of post translation modification transform the precursor peptide into the mature natural product. One of the common types of modifications observed in RiPP natural products is cyclization. My work in the RiPP natural product field has focused on three types of cyclization. In the first, thiazole and oxazole heterocycles are observed to be formed through the condensation of a serine, threonine, or cysteine residue and its backbone carbonyl in several classes of RiPPs including cyanobactins, thiopeptides, and linear azole containing (LAP) peptides. In Chapter 2, I discuss the crystallization of the putative cyclodehydratase YcaO. My structural study of YcaO allowed for the identification and characterization of new ATP binding motif present in this family of enzymes. Head to tail macrocyclization is a second type of cyclization observed in serveral RiPP classes including orbitides, cyanobactins, and cyclotides. In Chapter 3, I discuss the structural and biochemical characterization of PCY1, the enzyme responsible for cyclization of the orbitide segetalin A1. My work identified both a new example of a RiPP precursor peptide binding mode and the mechanistic basis for the new cyclization activity discovered in the abundant S9 peptidase fold. Finally, the cyclization of the N-terminus of a peptide and a side chain through an isopeptide bond is observed in lasso peptide class. These natural products are noteworthy because the tail of the lasso peptide is threaded through the macrocyclic structure, producing a lasso shape. In Chapter 4, I discuss the structural elucidation and biochemical characterization of the leader peptide binding domain TbiB1. Though the use of in vitro activity assays and binding experiments, I was able to demonstrate a short, conserved motif is both necessary and sufficient for binding. In Chapter 5, I elaborate upon my work on the lasso peptide isopeptidase AtxE2. This enzyme, theorized to be a siderophore or mechanism of self-resistance in the lasso peptide-producing organism, was studied both structurally and biochemically to demonstrate that it constitutes a previously uncharacterized fold. Furthermore, its sparse interactions with the lasso peptide substrate may help to explain its substrate tolerance. Finally, we use the AtxE2 protein sequence as a tool to identify additional putative lasso peptide biosynthetic clusters, several of which would have been difficult to identify using other proteins as a probe. In addition to the production of natural products, enzymatic tools have been actively sought in the product of biofuels. One of the rate limiting steps in the production of biofuels in the degradation of complex sugar polymers such as hemicellulose and lignin from plant biomass into simple sugars. These sugar monomers can be utilized as a carbon source for biodiesel or ethanol producing microbes. In Chapters 6 and 7, I detail my efforts to structurally characterize several types of sugar binding domains present within sugar hydrolases. The structural analysis was then augmented with binding assays to confirm the binding determinants.

Graduation Semester

2016-12

Type of Resource

text

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Copyright and License Information

Copyright 2016 Jonathan Chekan

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