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PMID:9395452

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Citation

Nagy, JM, Cass, AE and Brown, KA (1997) Purification and characterization of recombinant catalase-peroxidase, which confers isoniazid sensitivity in Mycobacterium tuberculosis. J. Biol. Chem. 272:31265-71

Abstract

The Mycobacterium tuberculosis katG gene encodes a dual-function enzyme called catalase-peroxidase, which confers sensitivity in M. tuberculosis to isonicotinic acid hydrazide. We have constructed a system for the high level expression of a recombinant form of this enzyme by amplifying the katG gene from the pYZ56 construct (1) and subcloning into a vector suitable for expression in Escherichia coli. The resulting plasmid, pTBCP, produced the catalase-peroxidase in large quantities, corresponding to 30% of total cell protein. The enzyme has been purified to homogeneity and appears to be a dimer in the native form. Using either hydrogen peroxide or t-butyl hydroperoxide and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as substrates, kcat and Km values have been obtained for both catalatic and peroxidatic activities, respectively. The availability of significant quantities of an active, folded, recombinant form of M. tuberculosis catalase-peroxidase should thus facilitate future studies of its role in drug activation and antibiotic resistance.

Links

PubMed

Keywords

Bacterial Proteins; DNA, Bacterial/chemistry; Escherichia coli; Isoniazid/pharmacology; Kinetics; Microbial Sensitivity Tests; Mycobacterium tuberculosis/enzymology; Mycobacterium tuberculosis/genetics; Peroxidases/chemistry; Peroxidases/genetics; Peroxidases/isolation & purification; Peroxidases/metabolism; Potassium Cyanide/metabolism; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

MYCTU:KATG

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P9WIE5

F

Seeded From UniProt

complete

MYCTU:KATG

involved_in

GO:0042744: hydrogen peroxide catabolic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

MYCTU:KATG

enables

GO:0004096: catalase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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