STRUCTURE AND FUNCTION OF CATALASE-PEROXIDASE (KATG) FROM MYCOBACTERIUM TUBERCULOSIS ISONIAZID RESISTANCE

• Main Author: Astuti, Winni
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32817
• Institution: Institut Teknologi Bandung

The catalase-peroxidase (KatG) from Mycobacterium tuberculosis has gained scientific interest due to its function in activating isoniazid (INH), an antitubercular drug. KatG is a homodimer of N-terminal domain, possessing heme-group and a bifunctional active-site, and C-terminal domain with unclear function. A clinical isolate of M. tuberculosis L10 exhibited resistance to INH at 1 ?g/mL and has no mutation at codon 315, the most abundant mutated codon in katG. The L10 M. tuberculosis clinical isolate was identified with new mutations in the katG gene, namely, T1310C, G1388T, G1481A, T1553C, and A1660G, which correspond to amino acid substitutions of Leu437Pro, Arg483Leu, Gly494Asp, Ile518Thr, and Lys554Glu, respectively. All of the amino acid substitutions were the conserved amino acid residues and were found in the C-terminal domain. The L10 M. tuberculosis clinical isolate showed a significant effect on the catalase-peroxidase activity. However, it has not yet cleared which mutations have the greatest effect on the catalase-peroxidase activity and INH activation. In the present study, the roles of KatG C-terminal domain amino acids that were mutated in the KatG of INH-resistant M. tuberculosis (Leu437Pro, Gly494Asp, Ile518Thr, and Lys554Glu) were investigated using site-directed mutagenesis and kinetic analysis. All purified KatG mutants exhibited a similar heme environment compared to that of the wild-type KatG (wtKatG). These results suggest that mutations at the positions Leu437, Gly494, Ile518, and Lys554 have no effect on disrupting the heme group in the N-terminal domain. However, the kcat/KM catalase values of the Leu437Pro, Gly494Asp, Ile518Thr, and Lys554Glu mutants were 2.1-, 10-, 6.1-, and 2.2-fold lower, respectively, than that of wtKatG. Meanwhile, the catalytic efficiency of the peroxidase dropped to 60, 45, 40, and 30% for the Leu437Pro, Gly494Asp, Ile518Thr, and Lys554Glu mutants, respectively. Indeed, the ability of the enzymes to activate INH by forming an IN-NAD complex declined to 22, 78, 78, and 57% for the Leu437Pro, Gly494Asp, Ile518Thr, and Lys554Glu mutants, respectively. Structural models of the Leu437Pro, Gly494Asp, Ile518Thr, and Lys554Glu KatG mutants shared a similar structure to that of wtKatG, with RMSD values of all atoms of 0.548 Å. Each KatG mutant contains local conformational changes around the position of the mutation. In addition to the local structure, each of the amino acid substitutions in the C-terminal domain may trigger structural changes of the substrate-binding pocket in the N-terminal domain such as residues Asp137, Ser315, and Arg418. Single mutation of the conserved amino acids (Leu437Pro, Gly494Asp, Ile518Thr, or Lys554Glu) in the C-terminal domain of KatG reduced the catalytic efficiency of its catalase and/or peroxidase activity, as well as its ability to activate the prodrug INH. It is also reasonable to propose that Gly494 is the most essential amino acid residue to direct the formation of catalytically active KatG, particularly for the catalase and INH activation activities. In the future, the function of the conserved amino acid residue needs to be examined whether it serves as a folding platform for KatG or it has more of a domino effect on the key amino acid residues for KatG catalysis. Hence, further investigations of the crystallographic structure, as well as studies using other approaches, are necessary.