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

Anuradha Gopal Bhat, Majety Naga Leelaram, Shivanand Manjunath Hegde, Valakunja Nagaraja

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Mycobacterium smegmatis topoisomerase I (MstopoI) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage in the absence of Mg2+, but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+ in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike MstopoI and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.

Original languageEnglish (US)
Pages (from-to)788-802
Number of pages15
JournalJournal of Molecular Biology
Volume393
Issue number4
DOIs
StatePublished - Nov 6 2009
Externally publishedYes

Fingerprint

Mycobacterium smegmatis
Type I DNA Topoisomerase
Metals
DNA Cleavage
Enzymes
DNA Primase
Escherichia coli
Type II DNA Topoisomerase
Poisons
Single-Stranded DNA
DNA
Zinc Fingers
Mutagenesis
Alanine
Bacteria
Mutation

Keywords

  • DNA relaxation
  • metal coordination
  • mycobacteria
  • topoisomerase I
  • toprim domain

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I. / Bhat, Anuradha Gopal; Leelaram, Majety Naga; Hegde, Shivanand Manjunath; Nagaraja, Valakunja.

In: Journal of Molecular Biology, Vol. 393, No. 4, 06.11.2009, p. 788-802.

Research output: Contribution to journalArticle

Bhat, Anuradha Gopal ; Leelaram, Majety Naga ; Hegde, Shivanand Manjunath ; Nagaraja, Valakunja. / Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I. In: Journal of Molecular Biology. 2009 ; Vol. 393, No. 4. pp. 788-802.
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