Purification and characterization of recombinant catalase-peroxidase, which confers isoniazid sensitivity in Mycobacterium tuberculosis.
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.
Nagy
J M
JM
Department of Biochemistry, Imperial College of Science Technology and Medicine, Exhibition Road, London SW7 2AY, United Kingdom.
Cass
A E
AE
Brown
K A
KA
D001426:Bacterial Proteins; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli; D007538:Isoniazid / Q000494:pharmacology*; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011190:Potassium Cyanide / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010544:Peroxidases; C076136:catalase HPI; D011190:Potassium Cyanide; D007538:Isoniazid