Studies on the Catechol Dioxygenases

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of the analogues have been simplified from a cyclohexadienone to a cyclohexanone and the hydroperoxide group has been replaced by either a hydroxymethyl or hydroxyethyl group.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 (K_i 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.