Investigations into the biosynthesis and mode of action of the phosphonate antibiotic dehydrophos

Circello, Benjamin T.

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https://hdl.handle.net/2142/18576 Copy

Description

Title

Investigations into the biosynthesis and mode of action of the phosphonate antibiotic dehydrophos

Author(s)

Circello, Benjamin T.

Issue Date

2011-01-21T22:47:11Z

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

Metcalf, William W.

Doctoral Committee Chair(s)

Metcalf, William W.

Committee Member(s)

• Farrand, Stephen K.
• Olsen, Gary J.
• Slauch, James M.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Date of Ingest

2011-01-21T22:47:11Z

Keyword(s)

• phosphonate
• antibiotic
• Streptomyces
• drug discovery

Abstract

Dehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad spectrum antibiotic activity. Dehydrophos antibiotic activity was determined to be dependent on uptake into the cell via non-specific oligopeptide permeases, followed by aminopeptidase catalyzed digestion. Bioactivity was abolished in Salmonella quadruple mutants lacking peptidases PepA, PepB, PepD, and PepN. However, reintroduction of any one of these peptidases resulted in restored bioactivity. Following peptidase action the released C-terminal portion of dehydrophos, a phosphonate analogue of dehydroalanine, undergoes a tautomeric rearrangement followed by hydrolysis to yield methyl acetylphosphonate, a structural analogue of pyruvate, and known inhibitor of pyruvate dehydrogenase and pyruvate oxidase. To identify genes necessary for the biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of Streptomyces lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments.

Graduation Semester

2010-12

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http://hdl.handle.net/2142/18576

Copyright and License Information

Copyright 2010 Benjamin T. Circello

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