Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

“…The mutations in these acidic residues lead to impairment in the catalytic activity and cytotoxicity (38,39), indicating that targeting the topoI through these residues may impair the enzyme function. In our earlier studies, we showed that the mutations of the individual amino acids, D108 and E112, of the motif in MstopoI to alanine resulted in 20-and 5-fold losses of activity, respectively (38), while mutations of the acidic residues in MttopoI led to complete loss of activity (25). In silico docking studies using the model of MttopoI indicated that imipramine and norclomipramine bind near the metal binding site of the enzyme (Fig.…”

“…The Mg 2ϩ binding mutants have been shown to be compromised in maintaining the cleavagereligation equilibrium, indicating that a disturbance in the metal coordination might abrogate the breakage and resealing mechanism of the enzyme. From these studies, we predicted that small molecules which target the metal binding motif might affect the enzyme activity (38). Indeed, in the oligonucleotide cleavage assay with the wild-type enzyme in the presence of the molecules, stimulation of the cleavage reaction was observed (Fig.…”

“…Earlier studies from our laboratory on the metal binding mutants of mycobacterial topoisomerase I revealed the role played by the triad of acidic amino acids within the TOPRIM domain in coordinating the Mg 2ϩ ion (25,38). The Mg 2ϩ binding mutants have been shown to be compromised in maintaining the cleavagereligation equilibrium, indicating that a disturbance in the metal coordination might abrogate the breakage and resealing mechanism of the enzyme.…”

“…Mg 2ϩ is an integral component in all topoisomerase reactions and is necessary for enzyme activity. The metal ion is not needed for the DNA cleavage but is important for the second transesterification reaction (38,52). The catalytic Mg 2ϩ is considered to be loosely bound with an ability to move within the enzyme (53).…”

“…The catalytic Mg 2ϩ is considered to be loosely bound with an ability to move within the enzyme (53). If indeed the catalytic Mg 2ϩ is a "moving metal ion" (38,53), the inhibitor interacting at the E112 residue would likely hinder such movement, arresting the reaction. High-resolution structures of the enzyme-inhibitor complex would validate such a mode of action.…”