Characterization of the S315G mutant of Mycobacterium tuberculosis catalase‐peroxidase KatG

Abstract

KatG(S315T) is the most common mutant found in clinical isolates of Mycobacterium tuberculosis resistant to the antibiotic Isoniazid (INH). Resistance due to the S315T mutation has been assigned to a decrease in the enzyme's affinity for the drug likely due to a decrease in the dimensions of a substrate access channel caused by the methyl group of the Thr 315 side chain. The main goal of the present study was to examine the effect of reducing the size of the side chain at residue 315 in this crucial region of the enzyme. Site-directed mutagenesis was used to prepare KatG(S315G). The overexpressed enzyme was characterized using optical and EPR spectroscopy. Catalase and peroxidase activities were reduced by nearly 2 fold and 10 fold, respectively, compared with the WT enzyme. Titration of KatG(S315G) with INH showed an approximately two-fold increase in affinity compared to WT KatG, while the rate of INH activation by KatG(S315G) was approximately 60% of that observed for the WT enzyme. Low temperature (10 K) EPR data showed the presence of mostly 5-c HS (high spin) and QS (quantum spin) heme spin states in the resting enzyme, more similar to KatG(S315T) than to WT enzyme. INH binding produced a transition from 6-c QS to 5-c QS and HS heme, also similar to the behavior of KatG (S315T). In addition, a small amount of 6-c low spin heme (due to the coordination of -OH group) was detected. The results are consistent with the hypothesis that a substrate access channel plays an important role in the binding of INH and its activation by KatG and that residue 315 plays a critical role in determining heme iron structure. Supported by NIH Grant # AI060014