| Catalase-peroxidases or KatGs are a class of multifunctional enzymes that in addition to their catalase and peroxidase activities also exhibit NADH oxidase, isoniazid lyase and isonicotinoyl-NAD synthase activities. Because of their role in the activation of isoniazid as an anti-tubercular drug, KatGs are the subject of intense study and the work in this thesis is focused on KatG from Burkholderia pseudomallei, BpKatG. BpKatG is 65% identical with KatG of M. tuberculosis (MtKatG), the enzyme responsible for the activation of isoniazid (INH), an anti-tuberculosis prodrug. MtKatG mutations are the most commonly found INH-resistance clinical isolates. The IN-NAD synthase activity is responsible for the synthesis of isonicotinoyl-NAD, the active form of INH. Previous work had confirmed that six residues on the distal side of the heme in KatGs including Arg108, Trp111, Tyr238, Met264, His112 and Arg426 were essential for catalase activity. The current work has extended these studies to residues in the main access channel including Asp141 and Ser324, which form the narrowest part of the main access channel and Glu242, ∼10 A from Asp141. In addition several BpKatG variants, equivalent to MtKatG variants found in INH-resistant clinical isolates of M tuberculosis, were investigated. Replacing Asp141 with a nonacidic residue significantly reduces catalase, but not peroxidase activity. It is proposed that binding of substrate H2O2 to Asp141 and Arg108 controls H2O 2 access to the heme active site. The Ser315Thr variant of MtKatG is the most commonly found mutation causing INH resistance. The equivalent variant in BpKatG, Ser324Thr, retains normal levels of catalase and peroxidase activities, whereas its NADH oxidase, INH lyase and isonicotinoyl-NAD synthase activities are reduced because the methyl group of Thr324, situated in the entrance channel to the heme cavity, creates a constriction or narrowing of the channel. Consequently, it is interference with INH accessibility that reduces IN-NAD synthesis. Replacing Glu242 with a nonacidic residue reduces catalase activity to 45% of native levels. Surprisingly, replacing the nearby residues G1n233 or Asn240 with an acidic residue causes an increase in catalase activity to three times that of native levels suggesting that the location of a negatively charged residue in the main access channel approximately 9-10 A from Asp141 is critical for enhancing the catalatic reaction. Changes in Asn142, hydrogen bonded to His112, causes decreased catalase and peroxidase activities and a decreased ability to activate isoniazid suggesting that the His-Asn hydrogen bond is critical and its absence may distort spatial organization of active-site residues resulting in changes in active site and/or binding site for H2O 2 and INH. The structural comparison studies of reverse mutations of BpKatG, Asn144Ser and Leu471Arg, with the equivalent mutations of MtKatG, Ser140Asn and Arg463Leu respectively show that the Ser140Asn and Arg463Leu mutations cause INH resistance most likely through conformational changes that result in less effective binding of INH and/or catalysis of INH lyase activity. Within the C-terminal domain of BpKatG, Leu594Met retains native catalase and peroxidase activities and native ability to activate INH. It is not clear how the Leu594Met mutation causes INH resistance. Expression of Leu626Pro was unsuccessful, indicating that stabilization of subunit-subunit interactions may be affected when Leu626 is replaced with a proline residue. Production of the Leu641Phe variant was successful at 28°C, but not at 37°C suggesting that Leu641Phe is a temperature-sensitive folding mutant, which explains how it causes INH resistance. |
