COMPUTATIONAL STUDY OF DIRECTED POINT MUTATION ON THE CATALYTIC RESIDUE OF HALOACID DEHALOGENASE FROM Pseudomonas aeruginosa ITB1 Nurul Nanda Khoiriyah, Enny Ratnaningsih, Ihsanawati
Department of Chemistry, Faculty of Mathematics and Natural Science, Institut Teknologi Bandung, Jalan Ganesa 10, Bandung 40132, Indonesia
Abstract
Organohalide used as pesticides are one of the dangerous pollutants because they are difficult to be decomposed and toxic to organism. Dehalogenase-producing bacteria are one of potential solution to restore organohalide contaminated areas because of its ability to catalyses the decomposition of organohalides into other non-toxic compounds. Pseudomonas aeruginosa ITB1 is a local bacterium that produces dehalogenase (named as Paed-d). This enzyme catalyses the degradation of monochloroacetic acid (MCA) to yield environmentally friendly hydroxy-ethanoic acid. In order to determine the tertiary structure of the Paed-d molecule and its interaction with MCA, this research was carried out using a computational method of directed point mutation on the catalytic site of Paed-d. The Paed-d tertiary structure obtained from the Swiss Model web service is the best structure selected for further analysis. Computational and literature studies on two groups of haloacid dehalogenase enzymes, namely group I and group II, indicated that Paed-d turns out to be a group II haloacid dehalogenase. Paed-d follows a homo-dimer structure with each monomer has a Rossmann-fold shaped core domain consisting of 6 β--sheet strands flanked by 5 α--helices with a cap domain consisting of 4 α--helical bundle. The Paed-d active site resides at the junction of the core domain and cap domain, with the D7 nucleophilic residue located at the end of the first β--sheet of the N-terminal end. Further analysis suggested that the Paed-d active site residues consist of D7, V8, F9, T11, R44, S121, N122, G123, K152, H178, and D181. The D7A mutation caused hydrogen bonds loss between MCA and D7, N122, and G123, and caused the Paed-d-MCA complex structure to become less stable, less compact, and more exposed to solvents. This could possibly cause Paed-d D7A to lose part or all of its activity as indicated by graph data of molecular dynamic simulation potential energy, radius of gyration, and SASA (Solvent-Accessible Surface Area). On the other hand, S121P mutation caused MCA to lose two hydrogen bonds with S121 and the Paed-d secondary structure changed from β--sheet to coil at residues 119-121 and 141-144. However, this S121P mutation did not cause a significant change in Paed-d-MCA complex structure. From the beginning, D14 did not act as a catalytic residue and was not proven to replace D7 as a nucleophile in Paed-d D7A because it was not resided in the catalytic pocket and was located far from the position of the MCA ligand. Finally, the catalytic mechanism was proposed based on the literature study and the results of this study. The result of computational studies is a potential first step to study the relationship between the structure and function of the haloacid dehalogenase.
