Crystallization and preliminary three-dimensional structure of the NahK/NahL: a protein complex involved in naphthalene metabolism

Full text: Many types of pollutants are released to the environment by anthropic activities related to the use of oil and its derivatives. Polycyclic aromatic hydrocarbons (PAHs) are an important family of such pollutants, some of which are mutagenic and carcinogenic. Several microorganisms were identified with the capability to metabolize these molecules, turning the bioremediation an interesting strategy for the reduction of PAHs from the environment. Naphthalene is a common and hazardous PAH, and the use of Pseudomonas putida for its degradation has been the focus of numerous studies. In P. putida G7, the enzymes NahK (4-oxalocrotonate decarboxylase) and NahL (vinylpyruvate hydratase) perform two sequential steps along the naphthalene degradation pathway. We present the cloning, expression, purification, crystallization assays and X-ray analysis of the NahK/NahL complex. The complex was also subjected to Circular Dichroism for secondary structure analysis; Dynamic Light Scattering and Small-Angle X-ray Scattering for oligomeric state characterization. These results were ambiguous about the oligomeric state of the complex, suggesting a hexamer or an octamer assembly. This doubt was solved by the crystal structure of the complex at 2.5 Å resolution, with unit-cell parameters a=100.41, b=219.96, c=310.36 and space group p212121, where two heterodecamers were identified. The structure was solved by the Molecular Replacement method and further structural refinement is currently underway. (author)