IN SILICO STUDIES ON THE INTERACTION OF HALOACID DEHALOGENASE FROM KLEBSIELLA PNEUMONIAE ITB1 (HAKP1) TOWARDS L-2- CHLOROALKANOIC ACID

• Main Author: Khansa Akmal, Meutia
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/57352
• Institution: Institut Teknologi Bandung

Organohalogens are organic compounds that have covalent bonds between carbon and halogens. Most of the organohalogen wastes are pollutants, harmful to the environment because they are difficult to be decomposed and toxic to otganisms. Organohalogens, particularly haloacids, can be enzymatically degraded by haloacid dehalogenase (HAD). Based on the catalytic mechanism, there are two types of HAD, namely HAD type I and HAD type II. In contrast to type II HAD, dehalogenation by type I HAD does not involve the formation of enzyme-substrate intermediates because the water molecules directly attack the C-2 substrate. The haloacid dehalogenase gene from Klebsiella pneumoniae ITB1 (called hakp1) has been isolated and cloned into pET-30a(+) and expressed in E. coli BL21 (DE3). This study aims to investigate the interaction of Hakp1 with L-2-chloroalkanoic acid substrate with variations on the length of carbon chain (C2-C5) and the amount of chlorine substituents on the C-2 substrate (mono- and di-) in silico with molecular docking. Modelling of Hakp1 with multiple methods and servers provides five tertiary structural models. Evaluation of these five structures shows that the best Hakp1 tertiary model was obtained from the Robetta server (TrRefineRosetta). Sequence alignment, tertiary structure alignment, and secondary structure analysis of Hakp1 toward HAD type I and HAD type II indicated that Hakp1 can be classified as HAD type II. Active site determination of Hakp1 through literature study, analysis with servers, and alignment of Hakp1 with HAD type II suggested that Asp8, Thr12, Ser125, Lys159, and Tyr165 residues on Hakp1 play an important role in dehalogenation with Asp8 residue acts as nucleophile. Docking score analysis showed that the substrate affinity for Hakp1 increased with the increase in carbon chain length from C2 to C5 and the amount of chlorine on C-2 substrates from 1 to 2. This was predicted as more of Hakp1 residues could interact with the substrate in the increase of carbon chain length and the amount chlorine atoms on C-2 substrate. Conformation analysis of the substrate on the active site indicated that the substrate will become more bulky with the addition of carbon chain length or addition of chlorine substituent on the C-2 substrate. It could be concluded that the eight substrates could be bound to the active site of Hakp1 but monochloroacetic acid was the substrate that may be best catalyzed by Hakp1. This is because there is only one chlorine atom bonded to the C-2 substrate so that the structure is not bulky. Although the analysis suggested that these eight substrates can bind to the Hakp1 binding site, wet laboratory evidence is needed to confirm the occurrence of dehalogenation.