Genetic and Biochemical Analysis of Phosphinothricin Tripeptide Biosynthesis in Streptomyces Viridochromogenes
Blodgett, Joshua Alan Van Dyke
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https://hdl.handle.net/2142/86691
Description
Title
Genetic and Biochemical Analysis of Phosphinothricin Tripeptide Biosynthesis in Streptomyces Viridochromogenes
Author(s)
Blodgett, Joshua Alan Van Dyke
Issue Date
2007
Doctoral Committee Chair(s)
Metcalf, William W.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Language
eng
Abstract
Our investigations led to the discovery of new biosynthetic intermediates and revealed inaccuracies in the previously published pathway. Specifically, this thesis work established that step III of PTT biosynthesis is the reduction of phosphonoacetaldehyde to hydroxyethylphosphonate by PhpC, an alcohol dehydrogenase with a previously unrecognized role in PTT biosynthesis. We also demonstrated that step IV of PTT biosynthesis is the conversion of hydroxyethylphosphonate to hydroxymethylphosphonate in a chemically novel reaction catalyzed by PhpD, a protein with no recognizable homologs in GenBank. We found evidence to suggest that step V of PTT biosynthesis is the oxidation of hydroxymethylphosphonic acid to phosphonoformaldehyde by PhpE, an alcohol dehydrogenase. Phosphonoformaldehyde is likely then converted into the established intermediate phosphonoformate by the aldehyde dehydrogenase PhpJ (step VI). We demonstrated that phosphonoformate is activated by conjugation to CTP in a reaction catalyzed by PhpF that produces the previously undescribed intermediate CMP-5'-phosphonoformate. Finally, we presented evidence to suggest that CMP-5'-phosphonoformate is likely required for the net incorporation of phosphonoformate into carboxyphosphonoenolpyruvate. This body of work has important implications in our understanding of phosphonic acid antibiotic biosynthesis, a group with great therapeutic promise.
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