J Biol Chem, Vol. 275, Issue 6, 4112-4117, February 11, 2000

Mutational Analysis of Escherichia coli Topoisomerase IV
III. IDENTIFICATION OF A REGION OF ParE INVOLVED IN COVALENT CATALYSIS^*

Soon Bahng, Elena Mossessova^§, Pearl Nurse, and Kenneth J. Marians^§

From the  Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, and ^§ Molecular Biology Graduate Program, Cornell University Graduate School of Medical Sciences, New York, New York 10021

The products of three dominant-negative alleles of parE, encoding the ATP-binding subunit of topoisomerase IV (Topo IV), were purified and their activities characterized when reconstituted with ParC to form Topo IV. The ability of the ParE E418K, ParE G419D, and ParE G442D mutant Topo IVs to bind DNA, hydrolyze ATP, and close their ATP-dependent clamp was relatively unaffected. However, their ability to relax negatively supercoiled DNA was compromised significantly. This could be attributed to severe defects in covalent complex formation between ParC and DNA. Thus, these residues, which are far from the active site Tyr of ParC, contribute to covalent catalysis. This indicates that a dramatic conformational rearrangement of the protein likely occurs subsequent to the binding of the G segment at the DNA gate and prior to its opening.