Crystallographic studies of NahI and its mutants, an aldehyde dehydrogenase from naphthalene degradation pathway of Pseudomonas putida G7

Full text: Pseudomonas putida G7 is one of the most studied naphthalene-degrading species. Catabolic genes of NAH7 plasmid encodes for enzymes involved in the conversion of naphthalene to pyruvate and acetaldehyde. The NahI enzyme (2-hydroxymuconic semialdehyde dehydrogenase) is an aliphatic-metabolizing aldehyde dehydrogenase required for conversion of 2-hydroxymuconic semialdehyde to 2-hydroxymuconic acid. Crystals of recombinant NahI diffracted to 1.85 Å resolution and belong to the hexagonal space group P6422, with unit-cell parameters a = b =189.47 c = 79.28 Å. It is noteworthy that NahF (salicylaldehyde dehydrogenase), other enzyme of the same degradation pathway, is an aromatic-metabolizing ALDH but shares a common scaffold with NahI despite their overall low sequence identity and different substrate specificity. Since slight differences on catalytic site of these enzymes may be suggested to be responsible for their diverse kinetic properties within a variety of aldehyde substrates, three NahI mutants forms were proposed. Diffraction data were collected and the structure determination is still in progress by Molecular Replacement method. Kinetic characterization will be conducted in order to allow determination of kinetics parameters for a wide range of substrates and conditions. These data may give evidences for NahI reaction mechanism and substrate specificity and are particularly useful for enzyme engineering aiming enzyme-assisted degradation processes of pollutants compounds. (author)