DNA topoisomerase I from Mycobacterium smegmatis. An enzyme with distinct features.
A type I topoisomerase has been purified to homogeneity from Mycobacterium 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. Furthermore, the enzyme makes single-stranded nicks and the 5'-phosphoryl end of the nicked DNA gets covalently linked with a tyrosine residue of the enzyme. However, M. smegmatis enzyme shows some distinctive features from the prototype Escherichia coli topoisomerase I. The enzyme is relatively 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 tuberculosis topoisomerase I exhibits very similar properties with respect to size, stability, and reaction characteristics. Sequence comparison of topoisomerase I from E. coli and M. tuberculosis shows the absence of zinc-finger motifs in mycobacterial enzyme. Using a two-substrate assay system, we demonstrate that the enzyme acts processively at low ionic strength and switches over to distributive mode at high Mg2+ concentration. Significantly, the enzyme activity is stimulated by single strand DNA-binding protein. There is a potential to exploit the characteristics of the enzyme to develop it as a molecular target against mycobacterial infections.
Bhaduri
T
T
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Bagui
T K
TK
Sikder
D
D
Nagaraja
V
V
D003545:Cysteine / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D009161:Mycobacterium / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D004264:DNA Topoisomerases, Type I; D003545:Cysteine