DNA TOPOISOMERASE-I FROM MYCOBACTERIUM-SMEGMATIS - AN ENZYME WITH DISTINCT FEATURES

A type I topoisomerase has been purified to homogeneity from Mycobacte rium smegmatis, It is the largest single subunit enzyme of this class having molecular mass of 110 kDa. The enzyme is Mg2+ dependent and can relax negatively supercoiled DNA, catenate, and knot single-stranded DNA, thus having typical properties of type I topoisomerases, Furtherm ore, the enzyme makes single-stranded nicks and the 5'-phosphoryl end of the nicked DNA gets covalently linked with a tyrosine residue of th e enzyme. However, M. smegmatis enzyme shows some distinctive features from the prototype Escherichia coli topoisomerase I. The enzyme is re latively stable at higher temperatures and not inhibited by spermidine . It apparently does not contain any bound Zn2+ and on modification of cysteine residues retains the activity, suggesting the absence of the zinc-finger motif in DNA binding, Partially purified Mycobacterium tu berculosis topoisomerase I exhibits very similar properties with respe ct to size, stability, and reaction characteristics. Sequence comparis on of topoisomerase I from E. coli and M. tuberculosis shows the absen ce of zinc-finger motifs in mycobacterial enzyme. Using a two-substrat e assay system, we demonstrate that the enzyme acts processively at lo w ionic strength and switches over to distributive mode at high Mg2+ c oncentration. Significantly, the enzyme activity is stimulated by sing le strand DNA-binding protein, There is a potential to exploit the cha racteristics of the enzyme to develop it as a molecular target against mycobacterial infections.