Biochemical and synthetic chemical approaches to studying the biosynthesis and regulation of bacterial secondary metabolites

Kapoor, Iti

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https://hdl.handle.net/2142/102883 Copy

Description

Title

Biochemical and synthetic chemical approaches to studying the biosynthesis and regulation of bacterial secondary metabolites

Author(s)

Kapoor, Iti

Issue Date

2018-08-24

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

Nair, Satish K.

Doctoral Committee Chair(s)

Nair, Satish K.

Committee Member(s)

• Hergenrother, Paul J.
• Lu, Yi
• Gennis, Robert J.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

Roseoflavin, Avenolide, Protein crystallography

Abstract

For several decades, significant scientific interest has been dedicated to developing bioactive small molecules to treat pathogenic infections. Despite the huge amount of efforts in the past few decades, resistant strains continue to emerge with every introduction of new therapeutic compounds into the clinic, leading to multi-drug resistant (MDR) strains that continue to be a serious health problem. This implies an urgent need for novel antimicrobial drugs to be added to the plethora of the already existing ones to overcome resistance. Recent studies have depicted that the major contributing source of the antimicrobial compound library originates from natural products.1 Thus, research on their biosynthesis to understand their function is of the utmost importance to utilize nature’s tools to generate these bioactive small molecule variants. Bioactive natural products, the molecules produced by living organisms, which possess antibacterial, antiviral, antimalarial, cytotoxic, and/or immunosuppressive properties represent a plentiful source of medically relevant compounds. Despite the abundance of these molecules, their synthesis often involves major challenges due to their complex structures. However, nature has overcome several major problems that we encounter during the chemical synthesis of small molecules. For example, enzymes that act as biocatalysts by specifically binding and orienting small ligands for selective modifications have provided a solution for the challenging stereochemical and regioselective modifications on the starting material to produce a single product. For the past few decades, there has been an increasing interest and demand to utilize enzymes as an alternative method for the production of a variety of compounds for chemoenzymatic semi-synthesis. The high activity of enzymes under mild conditions and the absence of harsh chemicals and byproducts used in chemical synthesis further emboldens this approach. Enzymes can be utilized to avoid the expensive and time-consuming route of organic synthesis to produce bioactive molecules. Hence, an understanding of the mechanistic and molecular basis of enzymatic reactions may allow the design of catalysts that can generate novel compounds or can modify the existing pool of bioactive compounds to create more potent variants. Such studies also provide a platform for developing and designing small drug molecules that may regulate various biochemical pathways. This dissertation describes efforts to understand the catalytic mechanism of proteins involved in the biosynthesis of the medically active natural product roseoflavin (RoF) and studying the mechanism to understand the regulatory action of a small signaling molecule, avenolide, in avermectin production. The research projects described in this thesis provide a platform to approach the issue of antibiotic resistance biochemically. The research highlighted below focuses on natural products sourced from the different strains of the genus Streptomyces. The first part of the thesis represents the structural and biochemical characterization of a multifunctional enzyme, RosB, that is involved in the biosynthesis of RoF. RoF is the only known vitamin B2 analog, and as such has antibiotic activity against both Gram-positive and Gram-negative bacteria. It is biosynthesized from a vitamin B2 precursor via two major intermediates. RosB is a multifunctional enzyme that carries out a challenging transformation of an aromatic methyl group to the aromatic amine group in riboflavin-5’-phosphate (FMN). Understanding the mechanism of this transformation is the major goal of chapter 2. In this chapter, I discuss the structure-based approach we have followed to identify active site variants of RosB, a single enzyme that shows oxidoreductase, decarboxylase, and aminotransferase activity, and some of these variants stall the reaction at various points along the reaction coordinate. These structural studies allowed for an investigation into a plausible route for reaction progression and provide a molecular rationale for the mechanism of this unusual biocatalyst. In chapter 3, I discuss the binding of avenolide, a unique class of small signaling molecule that autoregulates the production of antibiotic avermectin in the industrial microorganism S. avermitilis through structural and biochemical characterization. My work in this chapter identified the new structural features of avenolide binding to its cognate receptor AvaR1 and antagonism of DNA binding by AvaR1 by the small molecule ligand. These studies also opened new opportunities to the genetic understanding of the regulatory mechanism for other microbial antibiotic production.

Graduation Semester

2018-12

Type of Resource

text

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Copyright and License Information

Copyright 2018 Iti Kapoor

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