Structure-function studies of the catalase-peroxidase BpKatG from Burkholderia pseudomallei

Burkholderia pseudomallei produces catalase-peroxidase (BpKatG) encoded by the katG gene as part of its cellular defence system against oxidative stress either as catalase or as peroxidase. BpKatG is 65% identical with KatG of Mycobacterium tuberculosis, the enzyme responsible for the activation of isoniazid, the anti-tubercular drug. A structure-function study of BpKatG was carried out via site-directed mutagenesis, focusing mainly on residues on the distal side of the heme active site. Changing the Arg108, His112 and Asp141 residues in BpKatG resulted in significant changes in enzymatic activities as well as changes in absorption spectra and susceptibility to inhibitors. Changes to His112 caused drastically decreased catalase and peroxidase activities, while changes to Arg108 lowered catalase and peroxidase activities, but not as much as changes to His112. These data have confirmed a catalytic role for His112 and Arg108 in compound I formation. Changes to Asp141 caused significantly decreased catalase activity when Ala and Asn were the replacing residues but not when Glu was the replacement. None of the replacements affected peroxidase activity. This result confirmed that Asp141 is involved in compound I reduction by H2O2, but not in compound I formation. Two mechanisms are presented to explain the involvement of Asp141 in compound I reduction, direct ionic interaction with the substrate and a possible electrical potential field effect.