Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site

Abstract

We have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme's multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drug target.

Rapid, direct detection of bacterial Topoisomerase 1-DNA adducts by RADAR/ELISA

“…4B, below). The limited toxicity may reflect structural differences between MtTop1 and EcTop1 (9) and/or physiological differences between E. coli and M. smegmatis .…”

“…4E). Adduct recovery was quantified by summing signals determined by densitometry of DNA-containing fractions (5-9) and normalizing to DNA concentration (Fig. 4F).…”

“…Efforts to identify potent small molecule inhibitors of bacterial Type IA enzymes have had only limited success thus far (7). Key to the religation step catalyzed by bacterial Top1 is coordination of a metal ion by aspartate residues of an acidic triad within the DxDxxG motif of the highly conserved Top1 TOPRIM domain (8,9) (Fig. 1B).…”

Mechanism and resistance for antimycobacterial activity of a fluoroquinophenoxazine compound

“…MtbTopI relaxation inhibition assay - MtbTopI was expressed in E. coli T7 Express crystal strain (New England Biolabs) and purified as previously described [13,14]. The relaxation activity of MtbTopI was assayed in a buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/ml gelatin, and 0.5 mM MgCl 2 .…”

A multifunctional type IA DNA/RNA topoisomerase with RNA hydrolysis and rRNA processing activities fromMycobacterium smegmatisandMycobacterium tuberculosis

“…Next, RNA religation activity (second transesterification reaction following strand passage) of the enzyme was assayed using a synthetic substrate in which the two ends of a linear RNA were brought into proximity using a short complementary DNA as an anchor (Materials and Methods; Figure 5C). The cleaved fragments seen in the reaction without Mg 2+ were religated by TopoI in a metal ion (Mg 2+ )-dependent manner ( Figure 5D), indicating Figure 6A; reaction path 1) (37,38). The presence of the 2'-hydroxyl group in RNA provides for an additional alternative cleavage mechanism ( Figure 6A; reaction path 2).…”