Mechanistic investigations of enzymes in phosphonate metabolism

Peck, Spencer

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https://hdl.handle.net/2142/73062 Copy

Description

Title

Mechanistic investigations of enzymes in phosphonate metabolism

Author(s)

Peck, Spencer

Issue Date

2015-01-21

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

van der Donk, Wilfred A.

Doctoral Committee Chair(s)

van der Donk, Wilfred A.

Committee Member(s)

• Mitchell, Douglas A.
• Metcalf, William W.
• Lu, Yi

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Phosphonate biosynthesis
• enzymology
• non-heme iron-dependent enzyme
• 2-Hydroxyethylphosphonate dioxygenase (HEPD)
• methylphosphonate synthase (MPnS)

Abstract

Synthetic and naturally-occurring phosphonates have found widespread use in both agriculture and medicine. A program at the Institute for Genomic Biology at the University of Illinois at Urbana-Champaign was established to discover novel phosphonate natural products. In addition to this effort, the metabolic pathways for the biosynthesis of these natural products as well as phosphonate catabolism were examined for interesting biochemical reactions. Herein are detailed my contributions toward this enterprise. 2-Hydroxyethylphosphonate dioxygenase (HEPD) is a non-heme iron enzyme that catalyzes the cleavage of the carbon-carbon bond of 2-hydroxyethylphosphonate (2- HEP) during the biosynthesis of the herbicide phosphinothricin. Mechanistic studies were undertaken to elucidate the mechanism of catalysis by HEPD. These studies demonstrated that an unusual iron(IV)-oxo intermediate is at the heart of the catalytic cycle of HEPD. Also reported in this dissertation are studies on an enzyme with distant homology to HEPD, methylphosphonate synthase (MPnS), which likewise breaks the carbon-carbon bond of 2-HEP. These results strongly suggested the possibility of a consensus mechanism between HEPD and MPnS. This hypothesis was further evaluated through a combination of substrate analog incubations, site-directed mutants, and 18O KIE studies. Additionally, this thesis presents a study on an alcohol dehydrogenase that reduces phosphonoacetaldehyde (PnAA) to 2-HEP; the implications for the biosynthetic pathway for fosfomycin are discussed. Finally, observations made while studying an aldehyde dehydrogenase that oxidizes PnAA to phosphonoacetate are reported.

Graduation Semester

2014-12

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http://hdl.handle.net/2142/73062

Copyright and License Information

Copyright 2014 Spencer C. Peck

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