Elucidation of the catalytic mechanisms of the non-haem iron-dependent catechol dioxygenases: synthesis of carba-analogues for hydroperoxide reaction intermediates

Abstract

The catalytic mechanisms of the non-haem iron-dependent intradiol and extradiol catechol dioxygenases are thought to involve transient hydroperoxide reaction intermediates, formed by reaction of a catechol substrate with dioxygen. The synthesis of carba-analogues of these intermediates is described in which the hydroperoxide functional group (–OOH) is replaced by a hydroxymethyl group (–CH₂OH), and the cyclohexadienone skeleton simplified to a cyclohexanone. Analogues of the “proximal” hydroperoxide in which the hydroxymethyl group was positioned axially with respect to the ring were found to act as reversible competitive inhibitors (K_i 0.7–7.6 mM) for the extradiol enzyme 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) from Escherichia coli, whereas analogues in which the hydroxymethyl group was positioned equatorially showed no inhibition. In contrast, assays versus the intradiol-cleaving protocatechuate 3,4-dioxygenase from Pseudomonas sp. showed inhibition only by an analogue containing an equatorial hydroxymethyl group (IC₅₀ 9.5 mM). These data support the existence of a proximal hydroperoxide intermediate in the extradiol catechol dioxygenase mechanism, and suggest that the conformation adopted by the hydroperoxide reaction intermediate may be an important determinant in the reaction specificity of the extradiol and intradiol dioxygenases.