Transformations of Phosphonates by Non-Heme Iron-Dependent Oxygenases
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Authors
Hupp, Kendall
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thesis
Language
eng
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Biochemistry , Enzymes , Chemistry
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Abstract
Inorganic phosphate (Pi) is an essential requirement of all life forms; however, Pi is not present in high abundance in the environment. This has driven microorganisms to evolve enzymes that cleave carbon-phosphorus (C-P) bonds that enable the use of organophosphonates (Pn) as an alternative source of Pi. The iron-dependent oxidative PhnY*/PhnZ pathway is known to convert 2-aminoethylphosphonic acid into glycine and Pi. Recent work by our lab has revealed that the homologs GmPhnY* and GmPhnZ1 from the marine bacterium Gimesia maris have a different substrate specificity, oxidatively transforming methylphosphonic acid into formic acid and Pi. We have used 31P NMR spectroscopy to determine enzyme activity for a variety of Pn substrates with GmPhnY* and GmPhnZ1. The EFI-EST server was used to create sequence similarity networks (SSNs) of PhnY* and PhnZ homologs to investigate how families of enzymes may correlate with substrate specificity. Based on the results from our SSNs we have carried out site directed mutagenesis to explore the molecular determinants of substrate specificity in PhnZ. Furthermore, we have investigated other clusters in the PhnZ SSN to find novel PhnZ enzymes which may have a broader substrate specificity.
The fungal metabolite Fosfonochlorin is a rare example of a halogenated organophosphonate (Pn) with an unknown biosynthetic origin. A gene cluster encoding Fosfonochlorin biosynthesis was identified in Fusarium and Talaromyces sp. genomes by the occurrence of phosphoenolpyruvate mutase and phosphonopyruvate decarboxylase genes which are known to occur in the biosynthetic pathways of many other Pns. Other gene products from this cluster have been selected for further study, including FfnC and FfnD. FfnC is shown to be a new example of an NADH-dependent aldehyde reductase that converts phosphonoacetaldehyde into 2-hydroxyethylphosphonic acid. FfnD is shown to be a non-heme iron / 2-oxoglutarate (Fe/2OG) dependent oxacyclase. It transforms 2-hydroxyethylphosphonic acid into epoxyethylphosphonic acid and can convert ethylphosphonic acid into 1-hydroxyethylphosphonic acid. SSNs and genome neighbourhood networks (GNNs) have been used to determine further potential gene candidates that may be present in the Fosfonochlorin biosynthetic pathway by identifying gene homology in clusters with known Fosfonochlorin producers.
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ProQuest PhD and Master's Theses International Dissemination Agreement
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Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.