Mechanistic investigations of enzymes in phosphonate metabolism

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Peck, Spencer

Contributors dc:contributor

• van der Donk, Wilfred A.
• Mitchell, Douglas A.
• Metcalf, William W.
• Lu, Yi

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

Statement dc:rights

• Copyright 2014 Spencer C. Peck

Language dc:language

en

Handle dc:identifier

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

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/73062