Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria

Leelaram, Majety Naga; Bhat, Anuradha Gopal; Nunna, Suneetha; Nagaraja, Valakunja; Manjunath, Ramanathapuram

doi:10.1016/j.tube.2009.05.004

Cited by 5 publications

(9 citation statements)

References 29 publications

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“…29 A homology model of MstopoI was built based on the well-studied EctopoI structure to understand the Mg 2+ coordination by the enzymes, which would account for the above observations. 8 The overlay structure for the NTD of MstopoI included all the catalytic domains along with the toprim domain (Fig.…”

Section: Mstopoi Model To Reveal Mg 2+ Coordinationmentioning

confidence: 99%

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

Bhat

Leelaram

Hegde

et al. 2009

Journal of Molecular Biology

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Section: Mstopoi Model To Reveal Mg 2+ Coordinationmentioning

confidence: 99%

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

Bhat

Leelaram

Hegde

et al. 2009

Journal of Molecular Biology

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“…From the panel of mAbs generated against MstopoI, 2 mAbs, 1E4F5 and 17B1IB4, reacted with the enzyme both in enzyme‐linked immunosorbent assay (ELISA) and immunoblotting (Supplemental Fig. S1 A, B ) and recognized aa 241–612 in the amino‐terminal region of the enzyme (25). Both the mAbs belonged to immunoglobulin G1 (IgG1) subclass (Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Protein was purified to near homogeneity by affinity and ion exchange chromatography, as described previously (27). Generation of mAbs against the protein has been described previously (25).…”

Section: Purification Of Mstopoi and Generation Of Mabsmentioning

confidence: 99%

“…S1D). The different affinities of the mAbs to the enzyme is expected, as they were obtained from 2 independent clones (25). To test their effect on the enzyme activity, the mAbs were preincubated with MstopoI, and the relaxation reaction was initiated with the addition of supercoiled pUC18 DNA to the binary complex.…”

Section: Inhibition Of Dna Relaxation Activitymentioning

confidence: 99%

“…To obtain inhibitors for mycobacterial topoI, we followed the successful strategy that we employed earlier for DNA gyrase of mycobacteria (24). A panel of monoclonal antibodies (mAbs) against Mycobacterium smegmatis topoI (MstopoI) was generated and characterized (25). We describe a novel mechanism of multistep inhibition of topoI from two different species of mycobacteria by mAbs.…”

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Type IA topoisomerase inhibition by clamp closure

Leelaram¹,

Bhat²,

Godbole³

et al. 2013

FASEB j.

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Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration. Unlike other inhibitors of topoisomerases, the mAbs inhibited several steps of relaxation reaction, namely DNA binding, cleavage, strand passage, and enzyme-DNA dissociation. The enhanced religation of the cleaved DNA in presence of the mAb indicated closing of the enzyme DNA gate. The formation of enzyme-DNA heterocatenane in the presence of the mAbs as a result of closing the gate could be inferred by the salt resistance of the complex, visualized by atomic force microscopy and confirmed by fluorescence measurements. Locking the enzyme-DNA complex as a closed clamp restricted the movements of the DNA gate, affecting all of the major steps of the relaxation reaction. Enzyme trapped on DNA in closed clamp conformation formed roadblock for the elongating DNA polymerase. The unusual multistep inhibition of mycobacterial topoisomerases may facilitate lead molecule development, and the mAbs would also serve as valuable tools to probe the enzyme mechanism.

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Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity

Godbole

Leelaram

Bhat

et al. 2012

Archives of Biochemistry and Biophysics

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Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria

Cited by 5 publications

References 29 publications

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

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

Type IA topoisomerase inhibition by clamp closure

Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity

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