TY - JOUR
T1 - Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I
AU - Bhat, Anuradha Gopal
AU - Leelaram, Majety Naga
AU - Hegde, Shivanand Manjunath
AU - Nagaraja, Valakunja
N1 - Funding Information:
We thank Y. C. Tse-Dinh and D. N. Rao for critical reading of the manuscript and T. Bhaduri, D. Sikder, P. Jain, and other members of the VN laboratory for the reagents and discussions. R. J. Deshpande and B. M. Arif from the Departments of Materials Engineering and Soil Mechanics, Indian Institute of Science (IISc), are acknowledged for assistance in AAS. We thank the Department of Biochemistry, IISc, for CD and fluorescence measurement studies. M. Selvaraj of the Department of Molecular Biophysics Unit, IISc, is acknowledged for his assistance in modeling mycobacterial topoisomerase I. M.N.L. is a recipient of Council of Scientific and Industrial Research Senior Research Fellowship, Goverment of India. This work is supported by grants from the Department of Science and Technology under Fast Track program (to A.G.B.) and the Indian Council of Medical Research (V.N.), by an Indo-Danish collaborative grant, and by a Centre of Excellence grant from the Department of Biotechnology (V.N.), Government of India. V.N. is a recipient of J. C. Bose fellowship of the Department of Science and Technology, Government of India.
PY - 2009/11/6
Y1 - 2009/11/6
N2 - 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.
AB - 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.
KW - DNA relaxation
KW - metal coordination
KW - mycobacteria
KW - topoisomerase I
KW - toprim domain
UR - http://www.scopus.com/inward/record.url?scp=70349795296&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=70349795296&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2009.08.064
DO - 10.1016/j.jmb.2009.08.064
M3 - Article
C2 - 19733176
AN - SCOPUS:70349795296
SN - 0022-2836
VL - 393
SP - 788
EP - 802
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -