Purification, Crystallization, and Modelling of Mutant Catalase-Peroxidase from Isoniazid Resistant Mycobacterium tuberculosis

• Main Author: Sebastian Tandra, Yanuar
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32231
• Institution: Institut Teknologi Bandung

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis (Mtb) is a bifunctional enzyme that has been shown to activate isoniazid (INH), a pro-drug that is important for antituberculosis treatment. KatG is encoded by a katG gene which could undergo mutations to cause a decrease in the KatG activity and cause INH resistancy in Mtb. The INH activation mechanism by KatG is still debated. Therefore, to understand the mechanism of INH activation, crystallographic studies of mutant KatGs are required. According to kinetic studies, one of the mutant KatG with the lowest activity of KatG was observed in which glycine (Gly) undergo substitution to aspartate (Asp) in residue 494. The objective of this research was to obtain protein crystals of KatG Gly494Asp using expression, purification, and crystallization approaches of the aforementioned enzyme. The katG gene which contained the mutation had been overexpressed in Escherichia coli MC1061. The expressed recombinant KatG was then purified using Co-NTA affinity chromatography, Sephacryl™ S-200 gel filtration, and Q-FF 16/10 HiPrep™ anion exchange chromatography. However, the purified protein constantly showed protein bands at around 80, 49, and 30 kDa in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Native-PAGE analysis of the same fraction showed one dominant band, indication for degradation of KatG. Crystallization solutions were screened on the purified mutant KatG but no crystal was observed. We have also modelled KatG Gly494Asp which resulted in similar overall structure to the wild-ype KatG (RMSD 1,62 Å for all atoms). The structural changes were observed in the N-terminal residues, which plays a role in KatG dimerization, as well as increased number of hydrogren bonds at residue 494 in this model compared to that of wild type KatG. These results suggest that Gly494Asp mutation might reduce the activity of KatG through destabilization of KatG dimer.