Reduced Affinity for Isoniazid in the S315T Mutant ofMycobacterium tuberculosis KatG Is a Key Factor in Antibiotic Resistance

Shengwei Yu,Stefania Girotto,Chiuhong Lee,Richard S Magliozzo

doi:10.1074/jbc.m300326200

Shengwei Yu, Stefania Girotto + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m300326200

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Abstract

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis is responsible for the activation of the antitubercular drug isonicotinic acid hydrazide (INH) and is important for survival of M. tuberculosis in macrophages. Characterization of the structure and catalytic mechanism of KatG is being pursued to provide insights into drug (INH) resistance in M. tuberculosis. Site-directed mutagenesis was used to prepare the INH-resistant mutant KatG[S315T], and the overexpressed enzyme was characterized and compared with wild-type KatG. KatG[S315T] exhibits a reduced tendency to form six-coordinate heme, because of coordination of water to iron during purification and storage, and also forms a highly unstable Compound III (oxyferrous enzyme). Catalase activity and peroxidase activity measured using t-butylhydroperoxide and o-dianisidine were moderately reduced in the mutant compared with wild-type KatG. Stopped-flow spectrophotometric experiments revealed a rate of Compound I formation similar to wild-type KatG using peroxyacetic acid to initiate the catalytic cycle, but no Compound I was detected when bulkier peroxides (chloroperoxybenzoic acid, t-butylhydroperoxide) were used. The affinity of resting (ferric) KatG[S315T] for INH, measured using isothermal titration calorimetry, was greatly reduced compared with wild-type KatG, as were rates of reaction of Compound I with the drug. These observations reveal that although KatG[S315T] maintains reasonably good steady state catalytic rates, poor binding of the drug to the enzyme limits drug activation and brings about INH resistance.

Highlights

Tuberculosis (TB)1 infection is the leading cause of death due to a single infectious agent, causing over 2 million deaths annually [1]
The KatG[S315T] protein used in these studies was produced in E. coli using an overexpression system carrying the mutated M. tuberculosis katG gene
A notable improvement in yield of holoenzyme was found after growth in the presence of ␦-ALA, though purified KatG[S315T] still had a low optical purity ratio compared with WT KatG

Summary

EXPERIMENTAL PROCEDURES

Materials—INH (Sigma-Aldrich) was re-crystallized from methanol before use. PAA (Sigma-Aldrich) (32%) was diluted to 10 mM in potassium phosphate buffer and was incubated with 780 units/ml catalase (Roche Molecular Biochemicals) for 4 h at 37 °C to remove hydrogen peroxide, followed by removal of the enzyme by ultrafiltration. The plasmid pKAT II (a gift from Stewart Cole, Institut Pasteur, Paris) was used as an overexpression vector for KatG [18] and as the source of the katG gene that was cloned into pKS IIϩ to generate pSY15 used for mutagenesis. The 1.0-kb ClaI-XhoI fragment of the katG gene was subcloned into the pKS IIϩ vector in two steps to generate pSY15 in which site-directed mutagenesis was performed. Data acquisition and analysis utilized Kinet-Asyst software (HiTech Scientific), and all reactions were performed in potassium phosphate buffer, pH 7.2, at 25 °C, as described by Chouchane et al [20]. Reliable titration data were obtained using 20 ␮M enzyme in the sample cell (1.4 ml) and an INH concentration in the ligand delivery syringe that gave an INH-to-heme ratio of either 1.4:1 or 30:1 (for KatG[S315T] only) at the end of the titration. MODEL1000 chemical-freeze quench apparatus according to a published procedure [21]

RESULTS

WT KatG

DISCUSSION