Abstract
Extradiol-cleaving catechol dioxygenases function by binding both the organic substrate and O2 at a divalent metal center in the active site. They have proven to be a particularly versatile group of enzymes with which to study the O2 activation process. Here, recent studies of homoprotocatechuate 2,3-dioxygenase are summarized, showing how nature can utilize the enzyme structure and the properties of the metal and the substrate to select among many possible chemical paths to achieve both specificity and efficiency. Possible intermediates in the mechanism have been trapped by swapping active-site metals, introducing active-site amino acid substituted variants, and using substrates with different electron-donating capacities. Although each of these intermediates could form part of a viable reaction pathway, kinetic measurements significantly limit the likely candidates. Structural, kinetic, spectroscopic, and computational analyses of the various intermediates shed light on how catalytic efficiency can be achieved.