Datensatz

Zusammenfassung

Catecholate dioxygenases catalyse C-C bond cleavage and ring opening of catecholates, assisting in the degradation of aromatic compounds in soils (1,2). The aromatic ring of the substrate can be opened either between the two hydroxyl groups, which is known as intradiol cleavage, or to one side, known as extradiol cleavage. Intradiol and extradiol enzymes show high specificity for intradiol or extradiol behaviour. Understanding the factors which control the selectivity for intradiol or extradiol behaviour is one of the most intruiging puzzles in the study of dioxygenase reativity.
The active site of the extradiol enzyme consists of an iron or manganese centre in the M(II) oxidation state coordinated by two histidine ligands and a carboxylate group, a common motif in the non-heme family. Intradiol active sites in contrast have a two-histidine, tyrosine binding mode and utilise an Fe(III) active site. Although it would be tempting to ascribe the enzyme selectivity to the differences in the active site and the metal oxidation state, a His200Asn mutant of the extradiol enzyme homoprotocatechuate 2,3-dioxygenase (HPCD) was found to perform intradiol ring opening of a modified substrate DHB (3). This was the first time that an extradiol enzyme had been induced to show intradiol behaviour.
Here we extent earlier studies on the mechanism (4) and use computational methods to undertake a detailed study of the electronic and environmental factors that control the selectivity of the extradiol enzyme homoprotocatechuate 2,3-dioxygenase (HPCD), through a careful exploration of the effects of the enzyme environment on the reactivity of the substrate. This has enabled us to uncover the electronic basis for the reactivity of the enzyme and the importance of the second sphere residues in controlling the selectivity of the reaction.