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Generation of a Zn2+ free oxygenase of phenol hydroxylase from Pseudomonas sp. strain CF600

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Generation of a Zn2+ free oxygenase of phenol hydroxylase from Pseudomonas sp. strain CF600

Khor, Natalie (2014) Generation of a Zn2+ free oxygenase of phenol hydroxylase from Pseudomonas sp. strain CF600. Masters thesis, Concordia University.

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Abstract

The phenol hydroxylase enzyme from Pseudomonas sp. strain CF600 is involved in the conversion of phenol to catechol, which subsequent enzymes break down to precursors for the Krebs cycle. The protein is a multi-component enzyme that is composed of reductase (DmpP), oxygenase (DmpLNO dimer), activator (DmpM) and assembly (DmpK) subunits. The active site, found in DmpN of the oxygenase component, has a binuclear iron centre required for activity. In DmpN, previous experiments have shown that there is also a Zn2+ ion coordinated by four cysteine residues, but the role that it plays in the enzyme has yet to be elucidated.
Site-directed mutagenesis was previously performed on two of the cysteine ligands in DmpN (C433A and C437A) that form a complex with Zn2+, but attempts to express and purify the variant proteins were unsuccessful. In this thesis, different techniques were used to attempt removal of Zn2+ from its binding site in the oxygenase. These techniques included dialysis and column buffer exchange in the presence of a combination of chelating agents, ethylenediaminetetraacetic acid (EDTA) or tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), reducing agents dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) and a denaturant, urea. Protein samples were analysed with circular dichroism and fluorescence spectroscopy for secondary and tertiary structure changes, respectively. In addition, inductively-coupled plasma mass spectrometry (ICP-MS) was used to determine the metal content of the treated proteins. The most successful approach was the combination of TPEN, DTT and urea followed by dialysis against metal-free buffer, where Zn2+ was reduced from 2.6 atoms per molecule in pre-dialysis to 0.1 atoms per molecule after dialysis. This treatment, however, also resulted in the removal of Fe2+ from the enzyme. The method developed for the preparation of the apo-enzyme will enable further experiments to investigate the roles of metal ions in this enzyme following their controlled re-insertion.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Khor, Natalie
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry
Date:April 2014
Thesis Supervisor(s):Powlowski, J.
ID Code:978515
Deposited By: NATALIE KHOR
Deposited On:30 Jun 2014 20:17
Last Modified:18 Jan 2018 17:47
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