Methods to enable the detection of targeted AmphDI glycosyltransferase mutants that transfer GDP-C2’epimycosamine to amphoteronolide

Sotelo, Anthony Elijah

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https://hdl.handle.net/2142/101369 Copy

Description

Title

Methods to enable the detection of targeted AmphDI glycosyltransferase mutants that transfer GDP-C2’epimycosamine to amphoteronolide

Author(s)

Sotelo, Anthony Elijah

Issue Date

2018-04-24

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

Burke, Martin D.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

• Non-toxic amphotericin B derivative
• amphotericin B, amphoternolide

Abstract

Invasive fungal infections remain a leading cause of morbidity and mortality in the world, accounting for ~1.5 million deaths each year. Currently available treatments have important limitations, and the emergence of pathogen resistance is further weakening the clinical efficacy of most of these medicines. Amphotericin B (AmB) remains the last line of defense in treating these infections. Remarkably, this potent and broad-spectrum fungicidal agent has evaded pathogen resistance despite clinical utilization for more than half a century. However, the clinical efficacy of AmB is limited by its extreme toxicity, particularly to the kidneys. Guided by our lab’s recent advances in understanding the primary mechanism by which AmB kills both yeast and human cells, we have identified a promising new derivative of this natural product: C2’epiAmB. This derivative is dramatically less-toxic than AmB in primary human renal cells, mice, and rats at the highest doses. With this exciting direction, efficient access to large amount of C2’epiAmB are necessary from both a discovery and application standpoint. Therefore, development of a chemoenzymatic synthesis to C2’epiAmB would be imperative. We set out to determine an effective strategy to detect an active glycotransferase using AmB as a test substrate. AmphDI is the reversible glycotransferase responsible for glycosylation of the aglycone acceptor AmdeB with GDP-mycosamine in the biosynthesis of AmB. Using the reverse glycosylation reaction, we identified robust detecting of deglycosylation product amphoternolide (AmdeB) using AmB as starting material. Unfortunately, C2’epiAmB was not accepted by AmphDI and would therefore require generating a large library of mutants to be screened. The deglycosylation reaction leads to low conversion, which contrasts with the 4-fold increase seen in the glycosylation reaction seen in previous reports. Importantly this glycosylation was used with GDP-mannose, the stereochemical surrogate of natural GDP-mycosamine. We decided to use the stereochemical surrogate of GDP-C2’epimycosamine, GDP-glucose, as the sugar donor to glycosylate AmdeB. Under our conditions, GDP-glucose like C2’epiAmB was not accepted by AmphDI. Therefore, an AmphDI enzyme that glycosylates AmdeB with GDP-glucose should also accept GDP-C2’epimycosamine. Using the same assay, the deglycosylation and glycosylation reactions should differentiate active and inactive AmphDI mutants.

Graduation Semester

2018-05

Type of Resource

text

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http://hdl.handle.net/2142/101369

Copyright and License Information

Copyright 2018 Anthony Sotelo

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