Use of site-directed mutagenesis to probe the structure, function and isoniazid activation of the catalase/peroxidase, KatG, from Mycobacterium tuberculosis

A series of mutants bearing single amino acid substitutions often encounter ed in the catalase/peroxidase, KatG, from isoniazid-resistant isolates of M ycobacterium tuberculosis has been produced by site-directed mutagenesis. T he resultant enzymes were overexpressed, purified and characterized. Replac ing Cys-20 by Ser abolished disulphide-bridge formation, but did not affect either dimerization of the enzyme or catalysis. The substitution of Thr-27 5, which is probably involved in electron transfer from the haem, by prolin e resulted in a highly unstable enzyme with insignificant enzyme activities . The most commonly occurring substitution in drug-resistant clinical isola tes is the replacement of Ser-315 by Thr; this lowered catalase and peroxid ase activities by 50% and caused a significant decrease in the KatG-mediate d inhibition of the activity of the NADH-dependent enoyl-[acyl-carrier prot ein] reductase, InhA, in vitro. The ability of this enzyme to produce free radicals from isoniazid was severely impaired, as judged by its loss of Nit roBlue Tetrazolium reduction activity. Replacement of Leu-587 by Pro result ed in marked instability of KatG, indicating that the C-terminal domain is also important for structural and functional integrity.