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

Cao, Nan; Tan, Kemin; Annamalai, Thirunavukkarasu; Joachimiak, Andrzej; Tse-Dinh, Yuk -Ching

doi:10.1093/nar/gky492

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

Journal Article · Thu Jun 14 00:00:00 EDT 2018 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gky492· OSTI ID:1491852

Cao, Nan ^[1]; Tan, Kemin ^[2]; Annamalai, Thirunavukkarasu ^[1]; Joachimiak, Andrzej ^[2]; Tse-Dinh, Yuk -Ching ^[1]

Florida Intl Univ., Miami, FL (United States)
Argonne National Lab. (ANL), Argonne, IL (United States)

Here, 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.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States)

Sponsoring Organization:: National Institutes of Health (NIH); Bill and Melinda Gates Foundation; USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1491852

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 14 Vol. 46; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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