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Studies on the Catechol Dioxygenases

Studies on the Catechol Dioxygenases
Studies on the Catechol Dioxygenases

The catalytic mechanism of 3-(2',3'-Dihydroxyphenyl) propionate 1',2'-Dioxygenase (MhpB) from Eschericia coli, a non-haem iron dependent extradiol dioxygenase, is thought to proceed either through transient C-1' or C-2' hydroperoxide intermediate in the reaction of 3-(2',3'-Dihydroxyphenyl) propionic acid with dioxygen. The diastereoselective synthesis of carba-analogues of the proposed intermediates is described herein. The carbon skeleton of the analogues have been simplified from a cyclohexadienone to a cyclohexanone and the hydroperoxide group has been replaced by either a hydroxymethyl or hydroxyethyl group. Compounds mimicking the putative C-2' intermediate, in which hydroxyalkyl substituents were positioned axially with respect to the cyclohexane ring, showed competitive inhibition (Ki 0.7-7.6mM) whereas those with equatorial hydroxyalkyl groups showed no inhibition. In contrast, an analogue with an equatorial hydroxymethyl group showed inhibition (IC509.5 mM) when assayed with protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas sp while those with axial hydroxymethyl groups showed no inhibition. The data is consistent with the existence of a C-2' hydroperoxide reaction intermediate in the extradiol catechol dioxygenase mechanism and suggests that the conformation of the intermediate in the active site is a determining factor in the specificity of the extradiol and intradiol enzymes.

University of Southampton
Winfield, Christopher John
590b92bc-448d-414a-ae7e-9882edb7b6f0
Winfield, Christopher John
590b92bc-448d-414a-ae7e-9882edb7b6f0

Winfield, Christopher John (2001) Studies on the Catechol Dioxygenases. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The catalytic mechanism of 3-(2',3'-Dihydroxyphenyl) propionate 1',2'-Dioxygenase (MhpB) from Eschericia coli, a non-haem iron dependent extradiol dioxygenase, is thought to proceed either through transient C-1' or C-2' hydroperoxide intermediate in the reaction of 3-(2',3'-Dihydroxyphenyl) propionic acid with dioxygen. The diastereoselective synthesis of carba-analogues of the proposed intermediates is described herein. The carbon skeleton of the analogues have been simplified from a cyclohexadienone to a cyclohexanone and the hydroperoxide group has been replaced by either a hydroxymethyl or hydroxyethyl group. Compounds mimicking the putative C-2' intermediate, in which hydroxyalkyl substituents were positioned axially with respect to the cyclohexane ring, showed competitive inhibition (Ki 0.7-7.6mM) whereas those with equatorial hydroxyalkyl groups showed no inhibition. In contrast, an analogue with an equatorial hydroxymethyl group showed inhibition (IC509.5 mM) when assayed with protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas sp while those with axial hydroxymethyl groups showed no inhibition. The data is consistent with the existence of a C-2' hydroperoxide reaction intermediate in the extradiol catechol dioxygenase mechanism and suggests that the conformation of the intermediate in the active site is a determining factor in the specificity of the extradiol and intradiol enzymes.

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Published date: 2001

Identifiers

Local EPrints ID: 464544
URI: http://eprints.soton.ac.uk/id/eprint/464544
PURE UUID: 489eb2b8-f265-4cab-8cf7-0ba77a22b623

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Date deposited: 04 Jul 2022 23:45
Last modified: 16 Mar 2024 19:35

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Author: Christopher John Winfield

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