The molecular basis for adaptive evolution in novel extradiol ...

Extradiol dioxygenase (EDO) catalyzes metal-dependent ring cleavage of catecholic substrates. We previously screened a metagenomic library of activated sludge used to treat industrial wastewater contaminated with phenols and cyanide to identify 43 EDO genes. Here, we have characterized the enzymes belonging to novel I.2.G, I.3.M and I.3.N subfamilies. The I.3.M and I.3.N EDOs were Fe(II) dependent and preferred bicyclic substrates, whereas the I.2.G EDOs were Mn(II) dependent, preferred monocyclic substrates and had the highest affinity for catechol reported thus far. The I.2.G EDOs were more tolerant against heat (60 degrees C for 1 h) and chemical inhibitors (H(2)O(2) and NaCN) than I.3.M and I.3.N EDOs. Considering the dominance of the I.2.G EDOs over all retrieved EDOs (20 of 43 clones) and the presence of cyanide in the environment, this high affinity for substrate and structural robustness should provide survival advantages to host microorganisms. The 20 I.2.G EDOs were classified into six groups based on the amino acid sequence of the predicted ancestor, 1A1. Enzymes were chosen from each group and characterized. Two descendents, 1D2 and 5B2, each had a k(cat)/K(M) approximately twofold higher than that of 1A1 and reduced thermal stability, suggesting that descendents of 1A1 have adapted evolutionarily by a trade-off of inherent stability for increased activity.