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Characterisation of phenol hydroxylase and its auxiliary proteins, DmpM and DmpK, from pseudomonas spl Strain CF600

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Characterisation of phenol hydroxylase and its auxiliary proteins, DmpM and DmpK, from pseudomonas spl Strain CF600

Cadieux, Elisabeth (2002) Characterisation of phenol hydroxylase and its auxiliary proteins, DmpM and DmpK, from pseudomonas spl Strain CF600. PhD thesis, Concordia University.

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Abstract

Phenol hydroxylase from Pseudomonas sp. strain CF600 is an enzyme comprising three components: DmpP, a reductase containing an FAD and a [2Fe-2S] cluster; DmpM, an activator protein required for efficient catalysis; and DmpLNO, an oxygenase containing a binuclear iron cluster. The oxygenase requires an accessory protein, DmpK, for diiron cluster assembly. This thesis describes the characterisation of DmpM, DmpK, and DmpLNO. DmpM was found to exist in two forms. DmpM purified from the native strain was active in stimulating phenol hydroxylase activity, whereas an inactive dimeric form accumulated when DmpM was expressed in E. coli . Various treatments readily activated dimeric DmpM. Dissociation of the dimeric form of DmpM was shown to precede denaturation at low protein concentrations resulting in activation. Assembly of the binuclear iron center in the oxygenase component of phenol hydroxylase was examined in the presence and absence of DmpK, a protein required for in vivo synthesis of active oxygenase. DmpK had an effect on reconstitution of apo-oxygenase only at lower ratios of Fe 2+ /apo-oxygenase and it accelerated reconstitution. DmpK did not stably bind Fe 2+ , and could not donate Fe 2+ in the presence of chelators, suggesting that its primary role is not to deliver Fe 2+ to the active site. When the binuclear iron site was occupied by Mn 2+ , DmpK and Fe 2+ were required for reconstitution. This suggests a role for DmpK in metal ion discrimination. The oxygenase is made up of three copurifying polypeptides, DmpLNO. Single turnover experiments confirm that DmpLNO contains the active site, but requires DmpM for efficient turnover. Mössbauer, EPR and UV/vis spectroscopy data, demonstrated the presence of two types of binuclear iron centers in DmpLNO. Treatment of the oxygenase with hydrogen peroxide and/or DTT was investigated in order to understand why a significant proportion of the enzyme was inactive. Site-directed and random mutagenesis were performed on DmpM to identify residues involved in its interaction with the oxygenase. Although mutation of six conserved residues activated phenol hydroxylase in steady-state assays, the double mutant M37T/V77A, resulting from random mutagenesis, did not activate phenol hydroxylase. These results suggest that multiple substitutions are necessary to affect DmpM's interaction with the oxygenase component.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Cadieux, Elisabeth
Pagination:xvi, 233 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Theses (Ph.D.)
Program:Chemistry and Biochemistry
Date:2002
Thesis Supervisor(s):Powlowski, Justin B
ID Code:1577
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:20
Last Modified:08 Dec 2010 10:21
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