SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING INVESTIGATION OF THE REACTION MECHANISM OF THE 4-OXALOCROTONATE DECARBOXYLASE – NAHK

Naphthalene is a toxic compound that can be metabolized by the bacterium Pseudomonas putida G7 due to the presence of the plasmid NAH7, which carries the necessary genes for this process. In this organism, the enzyme NahK (4-oxalocrotonate decarboxylase, 28.4 kDa) belongs to the lower meta-cleavage pathway and catalyzes the decarboxylation reaction of 4-oxalocrotonate to 2-hydroxy-2,4-pentadienoate and CO2 in the presence of the inorganic cofactor Mg2+. The objective of this work was to experimentally elucidate the action mechanism of the NahK enzyme by constructing mutants of the recombinant protein, altering the residues K64, K72, S164, F151, and F153 present in the active site to Alanine. These residues were kinetically and thermodynamically characterized. Additionally, a double mutation between K72A and S164A was constructed to determine its non-additivity based on substrate presence. Finally, the obtained data were compared to Molecular Dynamics simulations to assess the effect of individual residues on the stability of NahK. Mutant genes were commercially obtained, cloned into the pET28a-TEV vector, and expressed in Escherichia coli BL21(DE3) at 17 °C for 24 h. Cells were lysed, and the soluble fraction was purified. It was observed that all proposed mutants exhibited reduced activity compared to the native enzyme. Kinetic parameters indicated that the simultaneous removal of K72 and S164 and the double phenylalanine deletion nearly inhibited enzymatic activity, while the removal of S164 alone only reduced it. The calculation of ΔGbinding from fluorimetric assays corroborated the kinetic data, indicating that the double phenylalanine appear to be primarily responsible for proper substrate binding to the active site (in addition to the cofactor). This finding was also supported by Molecular Dynamics simulations. Furthermore, it was observed through Molecular Dynamics that the positively charged residues K64 and K72 have a significant impact on the reaction energy. These results contribute to the understanding of the previously proposed enzymatic mechanism, suggesting that some steps may be slightly different.